Subcellular localization and differential expression account for the function of the nematode resistance gene Hs4

Subcellular localization and differential expression account for the function of the nematode resistance gene Hs4 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Subcellular localization and differential expression account for the function of the nematode resistance gene Hs4 Annika Schildberg, Kevin Dorn, Christian Jung This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6653794/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Plant-parasitic nematodes are economically important threats to global crop production. The beet cyst nematode ( Heterodera schachtii ) is a crucial pest in sugar beet ( Beta vulgaris ). While all species of the genus Beta are highly susceptible, the three species of the beet wild relative genus Patellifolia are entirely resistant. Recently, we cloned the Hs4 gene from P. procumbens , which confers complete resistance. In this study, we aimed to determine whether putative Hs4 orthologs exist in Beta and Patellifolia species. The Hs4 gene consisted of 4999 bp, with six exons and five introns. Patellifolia species contain highly similar Hs4 homologs. Single nucleotide polymorphisms and insertions/deletions between the accessions and species could be detected. We found an exonic integration of three bases, resulting in the addition of one amino acid. Interestingly, this variant was present in single accessions of all three Patellifolia species. Beta vulgaris contains an Hs4 homolog ( BvHs4 ) with 60% protein identity to Hs4. BvHs4 homologs were present in all Beta species analyzed. Further, we examined the expression patterns of Hs4 and BvHs4 homologs. While Hs4 homologs from Patellifolia species are strongly expressed in roots, BvHs4 homologs are expressed mainly in leaves. When the spatio-temporal expression of Hs4 was examined, no response to nematode inoculation was observed. These results are highly relevant for searching for functional Hs4 alleles and breeding nematode-resistant varieties. Biological sciences/Genetics/Agricultural genetics Biological sciences/Genetics/Plant breeding Beta vulgaris Patellifolia Heterodera schachtii ER sugar beet plant parasitic nematodes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Sugar beet, fodder beet, garden beet, and chard are diploid species ( B. vulgaris ssp. vulgaris, 2n = 2x = 18) belonging to the first section of the genus Beta , together with the wild species B. macrocarpa , B. patula , B. vulgaris ssp. adanensis , and B. vulgaris ssp. maritima . The second section, Corollinae , comprises the Eastern Mediterranean and Southwestern Asian species B. corolliflora , B. intermedia , B. lomatogona , B. macrorhiza , B. nana , and B. trigyna (Romeiras et al. 2016 ).The genus Patellifolia was formerly described as Beta section Procumbentes Ulbr. but was revised to constitute a separate genus (Williams et al. 1976 ; Scott et al. 1977 ). It has diverged from the genus Beta around 25.3 mya (Romeiras et al. 2016 ). The genus forms the third gene pool and comprises the closely related diploid species P. procumbens and P. webbiana and the tetraploid P. patellaris (Frese et al. 2019 ; Sielemann et al. 2022 ). While cultivated beets have a narrow genetic makeup, the wild Patellifolia species are genetically heterogeneous and represent an important source of resistance against diverse pathogens like the Beet Necrotic Yellow Vein Virus (vectored by the fungus Polymyxa betae) and the beet cyst nematode (BCN) Heterodera schachtii (Panella et al. 2020 ). Therefore, the different species were employed extensively in crossing experiments with various Beta species, but the lack of chromosome homology and pronounced hybrid sterility severely hindered their use as genetic resources. Sugar beet is highly susceptible to BCN, which attacks the roots and induces a nutrient cell called syncytium (Sijmons et al. 1994 ; Wyss 2002 ). After passing through four larval stages, the maturing female forms a cyst filled with eggs. The syncytium is the only feeding source for the nematodes, and its development depends on its integrity. The BCN is the most significant pest in beet cultivation (Müller 1999 ; Daub 2021 ). There is no complete resistance within the Beta genus. In contrast, the Patellifolia species are entirely resistant to the pathogen. However, transferring the resistance genes is extremely difficult due to crossing barriers. After decades of research, only a few resistant beet lines have been selected carrying translocations from P. procumbens and P. webbiana (Savitsky 1975 ; Jung and Wricke 1987 ). Recently, Hs4 was cloned from a Patellifolia translocation attached to beet chromosome 9 (Kumar et al. 2021 ). Knocking out Hs4 turned the resistant sugar beet variety Nemata into a highly susceptible one. In contrast, Hs4 overexpression in a susceptible B. vulgaris background resulted in varying resistance depending on the gene expression level. Hs4 was predicted to be an Endoplasmic Reticulum (ER)-bound rhomboid-like protease of 210 amino acids. Most plant rhomboid proteases contain seven transmembrane domains (TMDs). They are characterized by a serine protease catalytic unit that cleaves the substrates inside the membrane. Rhomboid proteins can also lack the catalytic residues, rendering them inactive proteases, and are then classified as iRhoms (Freeman 2009 ; Urban and Dickey 2011 ). A B. vulgaris homolog (BvHs4) showed 60 % polypeptide similarity to Hs4. It differs from Hs4 by102 additional amino acids at the N-terminus, and it lacks the leader sequence that directs Hs4 to the ER. Therefore, differences in subcellular localization of the two homologs are highly likely. In this study, we aimed to determine whether functional orthologs of Hs4 exist in other Patellifolia and Beta species. We reasoned that the highly conserved structure of the Hs4-like proteins points to their function as BCN resistance genes in other species. We found highly similar Hs4 orthologs in all Patellifolia species, whereas BvHs4 -homologous genes in Beta species were highly divergent. Because all Beta species are susceptible, we reasoned that the lack of function could be due to the ectopic expression. Therefore, we determined the local expression of Hs4 homologs in a broad set of species in different tissues and under nematode infestation. We found that Hs4 was highest expressed in roots while BvHs4 expression peaked in leaves. The results suggest that Hs4 and its Patellifolia orthologs are the only functional nematode-resistance genes. These results shed new light on the function of Hs4 and have implications for breeding nematode-resistant Beta crops. Results Sequence variations between Hs4 and its homologs from Patellifolia and Beta species The published Hs4 sequence comprises 5664 bp with five exons and five introns (Kumar et al. 2021 ). This sequence contained a large stretch (964 bp) of unknown nucleotides in the first intron. We sequenced the unknown region to 107 bp, thereby reducing the genomic region of Hs4 to 4999 bp (Supplementary Fig. 1). The composition of the cDNA and the resulting protein remained unchanged. In the following, ' Hs4' refers to the 4999 bp long sequence. Using the primer combination AS_F7/AS_R7, we detected Hs4 amplicons in all P. procumbens , P. webbiana , and P. patellaris accessions analyzed. The P. procumbens , P. webbiana , and P. patellaris homologs were named PpHs4, PwHs4 , and PpatHs4 , respectively. Additionally, we screened the sugar beet translocation lines TR363 and TR520 and the hybrid variety Nemata with the same primer set. The primers also bind to the exons of the B. vulgaris Hs4 homolog (Fig. 1 ), so double bands could be observed after agarose gel electrophoresis (Fig. 2 ). The lower and upper bands are P. procumbens ( Hs4 , 1217 bp) and B. vulgaris ( BvHs4 , 1499 bp) amplicons. We expected genes identified from P. procumbens to exhibit the closest sequence similarity to Hs4 . Due to the close relatedness between P. procumbens and P. webbiana , we expected a high similarity between PpHs4 and PwHs4 sequences, while PpatHs4 should display less similarity to Hs4 . As a first result, Hs4 was conserved across all Patellifolia species. Apart from single nucleotide polymorphisms (SNPs) and short insertions/deletions (InDels) (Supplementary Table 3), the protein-coding exons 2–6 (Supplementary Fig. 3A) were highly conserved. The overall sequence identity was 95–99% (Table 1 ). As expected, the Hs4 sequences from the translocation lines ( Hs4 TR363/TR520 ) and Nemata ( Hs4 Nemata ) were 100% identical to Hs4 , thus confirming their origin. Moreover, PwHs4 sequences showed the highest identity to Hs4 (99%). Identity values between Hs4 on one side and PpHs4 and PpatHs4 were lower (95–99%) (Table 1 ). These sequences exhibited InDels extending the sequence by 29–30 bp (Table 1 ). Accordingly, most SNPs were shared between P. procumbens and P. patellaris accessions. Four InDels were located within intronic regions. Moreover, a three bp insertion (position 1302, exon 5) led to the addition of one amino acid (Supplementary Fig. 4, Supplementary Fig. 8, Supplementary Table 3). This insertion was present in four P. procumbens , two P. webbiana , and all P. patellaris accessions, as well as in the P. procumbens draft genome sequence. Table 1 DNA sequence identities of different Hs4 sequences compared to the Hs4 reference ORF of 2022 bp. The Hs4 homologs' suffix denotes the seed code or other identifiers. Similarities were calculated with the blastn suite (Camacho et al. 2009 ). Query sequence Length Identity [%] Gaps [%] Hs4 2022 100 0 PpHs4 100810 2051 95 1 PpHs4 100820 2052 95 1 PpHs4 100821 2032 97 0 PpHs4 IPK419 2022 99 0 PpHs4 IPK951 2025 99 0 PwHs4 100002 2022 99 0 PwHs4 100832 2024 99 0 PwHs4 100833 2022 99 0 PwHs4 IPK526 2025 99 0 PwHs4 IPK927 2025 99 0 PpatHs4 100012 2052 95 1 PpatHs4 930065 2052 95 1 PpatHs4 IPK10 2052 95 1 PpHs4 Draft 2025 99 0 Hs4 TR363 2022 100 0 Hs4 TR520 2022 100 0 Hs4 Nemata 2022 100 0 BvHs4 homologs are present in a broad set of Beta species. Based on the phylogeny of the genus Beta , we expected BvHs4 -like sequences in species of the genus Beta . We used the primer combination AS_F7/AS_R7 amplifying 1499 bp of BvHs4 to screen 14 Beta accessions (Fig. 2 B). As a result, amplicons of the expected size were visible in 12 accessions. The B. macrocarpa and B. patula amplicons were substantially shorter ( BmrHs4 , ca. 1300 bp; BpHs4 , ca. 1300 bp). B. corolliflora ( BcHs4 ) exhibited an additional amplicon ca. 1300 bp in size (Fig. 2 B). Sequencing the smaller B. corolliflora fragment revealed no homology to any known gene from B. vulgaris (data not shown), while the larger BcHs4 fragment was composed of different sequences. BcHs4 A was a perfect match to BvHs4 from the reference genome sequence, while another sequence ( BcHs4 B ) showed several SNPs, small InDels, and a prominent insertion of 97 bp (after 1522 bp of the BvHs4 RefBeet sequence) matching a BvHs4 homolog from the tolerant B. vulgaris genotype U2Bv. BcHs4 B and BvHs4 U2Bv displayed a 312 bp deletion (after 2713 bp of BvHs4 ) (Supplementary Fig. 5B). We then used a BvHs4 -specific primer combination (AS_BvHs4_F1/AS_BvHs4_R0, Fig. 1 ) for amplifying the whole BvHs4 open reading frame of 4342 bp from seven Beta accessions (Supplementary Fig. 6). Here, we found the 97 bp insertion and the 312 bp deletion to also be present in BpHs4 and BmrHs4 . Further, they contained the same small InDels found in BcHs4 B . BpHs4, BcHs4 B , and BvHs4 U2Bv shared single SNPs but not BmrHs4 (Supplementary Fig. 4). Excluding BmrHs4 , which shows exonic deviations of up to 15 bp, all sequence variations larger than three basepairs lie within intronic regions (Supplementary Fig. 3B, Supplementary Fig. 5B). In summary, the BvHs4 homologs from species of the genus Beta are highly similar (sequence identity values 97–100%), and the intron/exon structures are the same as in BvHs4 (Supplementary Fig. 3). None of the genes had higher similarities to Hs4 than BvHs4 (Table 2 ). In conclusion, none of the Beta Hs4 homologs will likely function as a nematode resistance gene. Table 2 DNA sequence identities of different BvHs4 homologs compared to the BvHs4 (4342 bp) and Hs4 (2022 bp) ORFs. The Hs4 homologs' suffix denotes the seed code or other identifiers. Similarities were calculated with the blastn suite (Camacho et al. 2009 ). compared to BvHs4 compared to Hs4 Query sequence Length Identity [%] Gaps [%] Identity [%] Gaps [%] BaHs4 4341 99 0 77 4 BcHs4 A 4343 99 0 76 4 BcHs4 B 4168 97 0 77 4 BmHs4 4347 99 0 77 4 BmHs4 Bmar1.0 4369 99 0 77 4 BmcHs4 4332 99 0 77 4 BmrHs4 960018 4166 90 3 76 4 BpHs4 4166 97 0 77 4 BpHs4 Bpat-1.0 4300 97 0 77 4 BvHs4 EL10_2 4334 99 0 77 4 BvHs4 W357B 4342 100 0 76 4 BvHs4 U2Bv 4165 97 0 77 4 Hs4 2022 76 4 - - Phylogenetic analysis indicates distinct clades of Hs4- and BvHs4-like polypeptides We compared the open reading frames of all Beta and Patellifolia accessions together with the rhomboid-like proteins of quinoa ( Chenopodium quinoa , LOC110702170 and LOC110705623), mung bean ( Vigna radiata , LOC106763922), and spinach ( Spinacia oleracea , LOC110775063) as outgroups. Apart from minor amino acid substitutions, the polypeptides from the Patellifolia species showed high conservation (Supplementary Fig. 8). A single amino acid insertion (after Hs4 position 132) compared to Hs4 was present in various accessions of all three Patellifolia species (Supplementary Fig. 7). The sequence motif 'LLRDRCPDN' was suggested to be Hs4-specific due to its absence from BvHs4 (Kumar et al. 2021 ). Surprisingly, it could only be found in four out of thirteen Patellifolia sequences, the translocation lines, and Nemata (Supplementary Table 4). Because all Patellifolia polypeptides carried two more amino acids at their 3’-end, which were absent from the Beta polypeptides, the conserved motif could be revised to 'CPDNKE'. The Beta polypeptides were highly similar to each other and to the outgroup proteins (Supplementary Fig. 8). The minor sequence variations of the BmrHs4 sequence may result in a non-functional polypeptide because of a stop codon at position 141 (Supplementary Fig. 8). Across the two genera, motives of up to fifteen amino acids (aa) were conserved. A prominent difference was the addition of roughly 100 aa at the N-terminus of all Beta proteins, resulting from an additional exon (Fig. 1 ). Then, we generated a maximum likelihood tree based on the polypeptide sequences (Fig. 3 ). The BvHs4 homologs clustered closest to the outgroup proteins, and the Patellifolia polypeptides were grouped further away. The Beta species formed a distinct clade, except for B. macrorhiza . The divergence point towards the Patellifolia polypeptides was supported by a high bootstrap value of 100, indicating that this clade is distinct from the Beta and the outgroup proteins (Fig. 3 ). Conclusively, the original Hs4 polypeptide grouped with the translocation lines and Nemata, which were all derived from the same P. procumbens source. Additionally, one PpHs4 and three PwHs4 polypeptides are part of that subclade. Interestingly, onePpHs4 and the remaining PwHs4 polypeptides formed a distinct subclade. The polypeptide derived from the P. procumbens draft genome was somewhat distantly related to Hs4. Unexpectedly, three P. procumbens accessions grouped with the P. patellaris accessions. Protein localization predictions underpin the diversification of Hs4 homologs across species of the genus Beta Hs4 was predicted to be localized within the ER membrane (Kumar et al. 2021 ) (Table 3 ). We analyzed the protein structure with DeepTMHMM (Hallgren et al. 2022 ), which predicts that Hs4 has six transmembrane domains (Supplementary Table 5). The added valine in PpHs4 100810 did not change its subcellular localization. Contrarily, the probability of an ER localization increased from 0.7973 for Hs4 to 0.8184 for PpHs4 100810 (Table 3 ). However, the additional valine led to a slight conformation change. Five amino acids assigned to a transmembrane domain in Hs4 were now predicted to be part of a loop located in the lumen, and hence, the proximate fourth transmembrane domain was slightly decreased in size (Supplementary Table 5). Table 3 Predicted localization and membrane association for Hs4 and its homologs from Patellifolia and Beta species compared to the A. thaliana rhomboid protease AtRBL11. The probability values were calculated using DeepLoc2.1 (Ødum et al. 2024 ). Hs4 PpHs4 100810 BvHs4 AtRBL11 Localization Prediction probability cytoplasm 0.0667 0.0695 0.0707 0.0796 nucleus 0.0761 0.0790 0.0566 0.0689 extracellular 0.1902 0.1712 0.0209 0.0218 cell membrane 0.1334 0.1330 0.0505 0.0623 mitochondrion 0.3080 0.2782 0.2748 0.3320 plastid 0.1721 0.2045 0.9158 0.9346 endoplasmic reticulum 0.7973 0.8184 0.2247 0.1656 lysosome/vacuole 0.4065 0.4335 0.0581 0.0521 Golgi apparatus 0.4089 0.4283 0.0673 0.0372 peroxisome 0.0274 0.0371 0.1004 0.1345 Membrane association peripheral 0.1530 0.1580 0.2850 0.3000 transmembrane 0.9670 0.9680 0.9580 0.9610 lipid anchor 0.0740 0.0770 0.0590 0.0550 soluble 0.1430 0.1450 0.1220 0.1130 BvHs4, in turn, is predicted to be a plastid-localized transmembrane protein exhibiting six transmembrane domains (prediction value 0.9158) (Table 3 ). The main conformation difference lies in its long cytosolic N-stretch, which is much shorter in Hs4 and PpHs4 100810 (Supplementary Table 5). Interestingly, using the AlphaFold Protein Structure Database (Varadi et al. 2022 ; Varadi et al. 2024 ), we found that Arabidopsis contains an Hs4 homolog with 57 % and 71 % polypeptide similarity to Hs and BvH4, respectively. The protein is classified as a rhomboid-like protease 11 (AtRBL11, UniProt identifier: Q84MB5) and, like BvHs4, located in the plastids (Table 3 ). It also has six transmembrane domains (Supplementary Table 4). Hs4 and its homologs are differentially regulated between Patellifolia and Beta species We measured the transcriptional activities of Hs4 and its homologs in Patellifolia and Beta species with and without nematode infection. The translocation lines and the Patellifolia species displayed up to 26.6-fold higher Hs4 expression in roots than in leaves (Fig. 4 A). Contrastingly, across all Beta species, the BvHs4 homologs showed up to 6x higher expression in leaves (Fig. 4 B). The leaf-to-root expression ratio was similar across the different species. Next, we analyzed the expression with and without nematode infection. After infection with H. schachtii , no females or cysts were observed on P. procumbens roots, whereas B. vulgaris roots were heavily infected. Female numbers ranged between 54 and 119 (mean: 83.4). P. procumbens showed the highest Hs4 expression in roots across all sampling points (Fig. 5 ). The root/leaf ratio did not differ much between non-inoculated and inoculated plants. Upregulation after nematode inoculation was not significant (Fig. 5 ). In the young sugar beet plants, BvHs4 expression was higher in roots than in leaves. However, shortly before inoculation, BvHs4 expression in leaves was 4.1x higher than in roots. The BvHs4 expression rates did not respond to nematode infection. Discussion In this study, we revised the genomic sequence of the nematode resistance gene Hs4 (Kumar et al. 2021 )We studied Hs4 homologs in various Patellifolia and Beta species and examined their expression in different tissues with and without H. schachtii infestations. Hs4 has been described to span 5664 bp (Kumar et al. 2021 ). After sequencing an unresolved region of 964 bp in the first intron, we found that Hs4 comprises 4999 bp. Kumar et al. ( 2021 ) claimed the gene to consist of five exons and introns each, disregarding the untranslated regions (UTRs) they had annotated (see Supplementary Fig. 1). Exons do not necessarily have to be protein-coding. Instead, UTRs are also considered exonic regions, though non-coding (Aspden et al. 2023 ). Hence, according to our studies, Hs4 consists of six exons and five introns. The Hs4 gene was predicted to encode a rhomboid protease (Kumar et al. 2021 ). According to Urban and Dickey ( 2011 ), the smallest catalytically active unit of rhomboid proteases consists of six transmembrane domains (TMD). These basic TMDs are mainly found in bacteria and partly in eukaryotes, though eukaryotes often have a seventh TMD (Urban and Dickey 2011 ). However, many eukaryotic rhomboids belong to the secretase subfamily, which can be divided into two clades: secretase-A rhomboids exhibit seven TMDs, and secretase-B rhomboids contain only the core region of six TMDs. Further, these two clades are differentiated by distinct sequences around the catalytic serine S106. In the secretase-A clade, proteins contain a highly conserved GxSxGVYA, while B-class secretases show a less stringent GxSxxxF sequence (Lemberg and Freeman 2007 ). When we analyzed Hs4 using DeepTMHMM, we found the protein to consist of six TMDs, which is conclusive in its classification as a rhomboid protease. Further, Hs4 (and all related proteins analyzed in the current study) harbors the GxSxxxF sequence, indicating its affiliation with the secretase-B clade. Species of the genus Patellifolia are highly resistant to H. schachtii (Golden 1959 ; Viglierchio 1960 ). According to our hypothesis, we detected highly similar homologs in all Patellifolia species. Nevertheless, sequence variations could be observed between and within the species. Sequences perfectly matching Hs4 were detected in the translocation lines and the hybrid variety Nemata, reassuring the origin of the Hs4 sequence. None of the Patellifolia accessions sequenced harbored a homolog with 100 % sequence identity to Hs4 . Hoever, PpHs4 IPK419 , PpHs4 IPK951 , and all PwHs4 homologs exhibited the highest similarity to Hs4 (99 %). Three base pairs were interated into exon 5, adding valine to the Hs4 protein present in all three species. However, we found that the additional amino acid changed neither the localization of Hs4 to the ER nor its TMD structure. The phylogenetic relationships among the different Patellifolia accessions have long since been discussed. While the diploid P. procumbens and P. webbiana were previously considered two extreme ecotypes of a single species (Curtis 1968 ; Wagner et al. 1989 ), recent phylogenetic analyses based on plastome data have pointed to the clear separation into two distinct albeit closely related species. The tetraploid P. patellaris is more distantly related to the two diploid Patellifolia species (Sielemann et al. 2022 ). In our study, two PpHs4 and all PwHs4 homologs showed high sequence similarity to Hs4 . Nevertheless, three PpHs4 and the PpatHs4 homologs showed several deviations from Hs4 . Considering the relationship between the Patellifolia species, similarities between P. procumbens and P. patellaris appear peculiar. However, though P. procumbens and P. webbiana are more closely related, the genus itself is very diverse. Depending on sampling locations, plant morphologies differ even within species (Frese et al. 2017 ). This was underpinned by molecular marker studies demonstrating high genetic diversity within P. procumbens (Nachtigall et al. 2016 ). Sequence variations between Hs4 homologs from different Patellifolia species have been recently described (Reeves and Richards 2022 ). Analyzing short haplotype loci polymorphic between different species, they identified 0.16–3.36 variants between Hs4 orthologs. Of these loci, 20 % contained single- or muti-nucleotide polymorphisms, and the remaining 80 % PAVs. Strikingly, Reeve and Richards (2022) detected differences in exonic variants at several Hs4 loci across accessions within the same species, which aligns with our results. In addition, we identified a major InDel in exon five of several Patellifolia accessions, resulting in an added valine, which had not been reported in the study by Reeves and Richards ( 2022 ). Instead, they reported four short haplotype loci with differing major variants in or around exon five, which, in turn, could not be detected in our study. Due to the high similarities between Hs4 and PwHs4 s from P. webbiana , we speculate that Hs4 might originate from P. webbiana . A second resistance gene is on chromosome 7 of P. procumbens and P. webbiana (Jung et al. 1986 ). Using the primers matching Hs4 , no other Hs4 homologous sequence could be amplified from the Patellifolia species, suggesting no Hs4 paralog on chromosome 7. The genera Patellifolia and Beta have diverged 25 mya (Romeiras et al. 2016 ). The general sequence similarity between sugar beet and Patellifolia genomes is 75 % (Reeves and Richards 2022 ). Apart from BvHs4 ,we detected a highly similar homolog in the wild progenitor of sugar beet, B. vulgaris ssp. maritima . As expected, the tetraploid species B. corolliflora (Sielemann et al. 2023b ) has two BvHs4 -homologous sequences that varied at several sites. While BcHs4 A resembled BvHs4 , BcHs4 B had a prominent insertion of 97 bp and a deletion of 312 bp, both of which were also present in B. patula and B. macrorhiza . Matching these results, B. macrorhiza is believed to be a parental species of B. corolliflora (Sielemann et al. 2023b ). The insertion is similar to the BvHs4 homolog from the nematode-tolerant sugar beet genotype U2Bv. This genotype is characterized as nematode tolerant based on the expression of two BvNLP7 genes that are located on chromosome 5 (Sielemann et al. 2023a ) and which do not show any resemblance to the nematode resistance gene Hs4 , whose Beta homolog is located on chromosome 2. Both BcHs4 variants lie in the intron, so they do not affect the resulting protein. Further, these sequences did not show higher similarity to Hs4 than BvHs4 and are not expected to contribute to nematode resistance. We were interested in the expression patterns of the Hs4 homologs from other species. Hs4 is highly expressed in roots and less in leaves. Noteworthy, we detected the highest expression levels in the translocation line TR520 and the hybrid variety Nemata derived from it. A second translocation line, TR363, did not show enhanced Hs4 expression levels. TR363 harbors a much smaller translocation than TR520 (Kumar et al. 2021 ), and important regulatory cis -elements might be lacking in its sequence, leading to differences in expression intensities. TR363 never gained importance in beet breeding due to its inferior yield and quality characteristics. BvHs4 , on the other hand, is expressed strongly in leaves. Its protein is predicted to target the chloroplast, similar to its Arabidopsis homolog AtRBL11 (Kmiec-Wisniewska et al. 2008 ). Its strikingly different expression pattern and low sequence homology to Hs4 indicate that BvHs4 and its homologs from other Beta species do not function as nematode-resistance genes. We speculate that Hs4 has acquired a new function in evolution, coupled with its localization in the ER membrane, which provides a typical example of the neo-functionalization of a gene. In conclusion, the significant structural differences between Hs4 and its Beta homologs and their different expression patterns prohibit a targeted modification of their function, e.g., by genome editing to convert them into resistance genes. Due to the poor agronomic performance of the nematode-resistant translocation lines, the expression of the Hs4 gene after transformation into sugar beet is the only realistic solution to breed resistant varieties. Materials and Methods Plant material and growth conditions Fifteen Beta and fifteen Patellifolia accessions, the resistant translocation lines TR363 and TR520, and the resistant variety Nemata derived from TR520 (Supplementary Table 1) were grown in a greenhouse or a climate chamber, respectively, under long-day conditions (LD; 16 h light / 8 h dark). DNA isolation and PCR Samples of different plant tissues were taken at several points, frozen in liquid nitrogen, and stored at -70°C until further usage. Following the grinding of the tissues, genomic DNA was isolated using the NucleoSpin Plant II kit (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions or the plant DNA mini preparation protocol (Dellaporta et al. 1983). Before further usage, the genomic DNA was checked on a 1% agarose gel (90V, 30 min or 80V, 12 min). A non-sequenced (unresolved) region in the first Hs4 intron was amplified from the variety Nemata (Supplementary Table 1) with a Taq polymerase (Biozym Scientific GmbH, Hessisch Oldendorf, Germany) using the primer combination AK_F2/AS_R5 (Supplementary Table 2). To amplify putative homologs of Hs4 and BvHs4 simultaneously, we designed a primer set (Fig. 1 ) binding to a DNA region encoding a highly conserved polypeptide sequence between Hs4 and BvHs4 (Supplementary Fig. 2). The primer pair AS_F7/AS_R7 was expected to amplify 1217 bp and 1499 bp of the Hs4 and BvHs4 genes (Supplementary Table 2). Taq polymerase (Biozym Scientific GmbH) or Phusion™ High-Fidelity polymerase (ThermoFisher Scientific™, Waltham, MA, USA) was used for amplifying the genomic DNA of Hs4 and its homologs from other species. The primer combination AK_F5/AK_R6 (Supplementary Table 2) was used for Hs4 ORF amplification whereas AS_BvHs4_F1/AS_BvHs4_R0 (Supplementary Table 2) was used to amplify the Beta homolog ORFs. The PCR products were analyzed on a 1% agarose gel (90V, 30–40 min). Plasmid cloning and Sanger sequencing Using the CloneJET PCR Cloning Kit (ThermoFisher Scientific™), DNA was cloned into the pJET1.2/blunt cloning vector according to the manufacturer's instructions and transformed into competent Escherichia coli (strain DH5α; DNA Cloning Service eK, Hamburg, Germany) via the heat-shock method (Froger and Hall 2007 ). Following overnight incubation at 37°C on LB plates supplemented with ampicillin (50 µg/ml), colonies were screened for the plasmid's presence by PCR. Positive colonies were cultured overnight in 5 ml LB with ampicillin on a shaker. Plasmids were then isolated using the NucleoSpin Plasmid QuickPure Kit (Macherey-Nagel) and used for PCR in a 1:1000 dilution. Amplified gene fragments or whole genes were Sanger sequenced on an Applied Biosystems 3730xl DNA Analyzer (ThermoScientific™, via IKMB, Kiel University) using a set of different primers (Supplementary Table 2). Phylogenetic analysis Sanger sequences were aligned to the reference sequences using the CLC Main Workbench 20 (Qiagen, Hilden, Germany). As a reference, the Hs4 gene sequence derived from TR520 by Kumar et al. ( 2021 ) was used for the Patellifolia sequences, and the genomic RefBeet-1.2.2 (Dohm et al. 2014 ) sequence of the rhomboid-like protein 11 from sugar beet (NCBI Reference Sequence XM_010669575.1) was deployed for all Beta sequences. After the alignment, specific Hs4 or BvHs4 sequences were generated for each accession sequenced. The CLC-incorporated tool was used for protein translation, generating the corresponding polypeptide sequences. Furthermore, we searched reference sequences from the susceptible beet lines EL10_2 (McGrath et al. 2022 ) and W357B v1.0 (Dorn 2022 ) and the nematode-tolerant B. vulgaris line U2Bv (Sielemann et al. 2023a ). Reference sequences from the B. patula accession Bpat-1.0 (Rodríguez del Río et al. 2019), the B. vulgaris ssp. maritima accession Bmar1.0 (Rodríguez delRío et al. 2019) and a first P. procumbens draft genome sequence (USDA_Ppro_WB292_v1.0, NCBI Genome Accession No. JBMGQN000000000, derived from USDA NPGS Accession Ames 4464). Using MEGA11 (Tamura et al. 2021 ), the sequences were aligned via the MUSCLE algorithm, and a maximum-likelihood phylogenetic tree (500 bootstraps) was generated. Alignments were visualized using pyBoxshade v. 1.2 (mdbaron42 2021 ). The blastn suite (Camacho et al. 2009 ) of the National Center for Biotechnology Information (Sayers et al. 2022) was deployed for DNA sequence comparisons. The localization and conformation of single polypeptides were predicted using DeepLoc2.1 (Ødum et al. 2024 ) and DeepTMHMM (Hallgren et al. 2022 ), respectively. In vivo nematode infection assay Nematode infection tests were performed essentially as described by Lange and De Bock ( 1994 ). The H. schachtii strain 'Schach 0' (Müller 1998 ) was propagated on susceptible sugar beet or rapeseed in sand-filled tubes in a greenhouse under LD conditions. Larvae were hatched from cysts in 3 mM ZnCl 2 in a Baermann-funnel apparatus. Before inoculation, the J2 larvae were concentrated to the desired density of 150 per ml. Plants were grown in plastic tubes filled with quartz sand and inoculated with 300 J2 larvae. Developing females were identified four weeks later. Expression analysis The leaf and root samples of three biological replicates per accession were taken for the initial expression test. For the spatiotemporal expression analysis, samples of plant roots (R), hypocotyls (H), cotyledons (C), and leaves (L) of three biological replicates were taken shortly after germination (R, H, C) upon the development of the first true leaves (R, C, L), 21 days after germination (R, L), before inoculation (R, L), and 3 and 28 days post-inoculation (dpi; R, L). The samples were frozen in liquid nitrogen, and total RNA was isolated using the Universal RNA Kit (roboklon, Berlin, Germany), according to the manufacturer's instructions. The RNA quality was checked using a NanoDrop2000 spectrophotometer (ThermoFisher Scientific™) and agarose gel electrophoresis (2% gel, 100V, 12 min). 100 ng of RNA were incubated with 1 U DNase (ThermoFisher Scientific™) at 37°C for 30 min. Subsequently, the DNase was inactivated by adding 1 µl EDTA (50 mM), followed by incubation at 65°C for 10 min. Then, the cDNA was generated using the First Strand cDNA Synthesis Kit (ThermoFisher Scientific™), following the manufacturer's instructions. Using the GAPDH primer combination for Beta (BvGAPDH_F/BvGAPDH_R, Supplementary Table 2) or Patellifolia (AS_BvPp_GAPDH_F/AS_BvPp_GAPDH_R, Supplementary Table 2) species, the cDNA quality was assessed by PCR. Following the manufacturer's instructions, two µl cDNA were used for quantitative reverse transcription PCR (RT-qPCR) deploying the Platinum™ SYBR™ Green qPCR SuperMix-UDG (Invitrogen™, ThermoFisher Scientific™). The GAPDH gene was used for gene expression normalization. For the detection of Hs4 transcripts, the primer combinations AK_F7/AK_R5 and AS_BvHs4_F1/AS_BvHs4_R7 (Supplementary Table 2) were used. The obtained Ct-values were evaluated using the Pfaffl method (Pfaffl 2001). Statistical analysis Normally distributed data were statistically analyzed by a one-way analysis of variance (ANOVA) using Microsoft Excel. The non-normally distributed data were statistically analyzed by the Kruskal-Wallis rank sum test, followed by a Hochberg-corrected Dunn post-hoc test using the online calculator Astatsa (Vasavada 2016 ) and the open-source statistical computing software R version 4.3.3 (R Core Team 2024 ) using the package "PMCMRplus" (Pohlert 2023 ). Declarations Data availability statement The P. procumbens sequence data generated and analysed during the current study are available in GenBank of the NCBI (https://www.ncbi.nlm.nih.gov/) repository (Genome Accession No. JBMGQN000000000). The Hs4 homologs sequences are available in GenBank of the NCBI (GenBank Accession Numbers PV258691 to PV258713) (Supplementary Table 6). Acknowledgments We thank Birgit Defant, Maike Schneider, S. Greve, and I. Baumgartner for their technical assistance. We thank the Institute of Clinical Molecular Biology in Kiel for providing Sanger sequencing, as partly supported by the DFG Cluster of Excellence "Inflammation at Interfaces" and "Future Ocean." Author contribution AS designed and performed the experiments, followed by analyzing the data. CJ led the design of the study and supervised data analysis. KD contributed unpublished sequence data. AS drafted the manuscript, which CJ and KD revised. All authors read and approved the final article. Correspondence and requests for materials should be addressed to CJ. Conflict of interest The authors declare no competing interests. References Aspden JL, Wallace EW, Whiffin N (2023) Not all exons are protein coding: Addressing a common misconception. Cell Genomics 3:100296. https://doi.org/10.1016/j.xgen.2023.100296 Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) BLAST+: architecture and applications. 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Cite Share Download PDF Status: Published Journal Publication published 28 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 09 Jul, 2025 Reviews received at journal 04 Jul, 2025 Reviews received at journal 23 Jun, 2025 Reviewers agreed at journal 16 Jun, 2025 Reviewers agreed at journal 15 Jun, 2025 Reviewers invited by journal 13 Jun, 2025 Editor assigned by journal 08 Jun, 2025 Editor invited by journal 27 May, 2025 Submission checks completed at journal 26 May, 2025 First submitted to journal 13 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6653794","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":471955989,"identity":"93827642-8865-4e47-aa2d-7f4bb0c8021c","order_by":0,"name":"Annika Schildberg","email":"","orcid":"","institution":"Christian-Albrechts-University of Kiel","correspondingAuthor":false,"prefix":"","firstName":"Annika","middleName":"","lastName":"Schildberg","suffix":""},{"id":471955990,"identity":"72069a7f-e095-4413-a8bd-cd7e5e96a3b3","order_by":1,"name":"Kevin Dorn","email":"","orcid":"","institution":"USDA","correspondingAuthor":false,"prefix":"","firstName":"Kevin","middleName":"","lastName":"Dorn","suffix":""},{"id":471955991,"identity":"1fc73cdb-1bea-4f4a-b5e2-5588fffb6f4b","order_by":2,"name":"Christian Jung","email":"data:image/png;base64,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","orcid":"","institution":"Christian-Albrechts-University of Kiel","correspondingAuthor":true,"prefix":"","firstName":"Christian","middleName":"","lastName":"Jung","suffix":""}],"badges":[],"createdAt":"2025-05-13 09:23:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6653794/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6653794/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-40666-5","type":"published","date":"2026-02-28T15:59:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84845673,"identity":"fe54c929-20c8-435f-bdc1-4714a14b5b12","added_by":"auto","created_at":"2025-06-18 03:00:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":50796,"visible":true,"origin":"","legend":"\u003cp\u003eStructure of the open reading frames (ORFs) of \u003cem\u003eHs4\u003c/em\u003e (2022 bp) and its \u003cem\u003eBeta\u003c/em\u003e homolog \u003cem\u003eBvHs4\u003c/em\u003e (4342 bp) and the intron-less coding sequences (CDS) derived from them. Exons are shown in green (\u003cem\u003eHs4\u003c/em\u003e) and orange (\u003cem\u003eBvHs4\u003c/em\u003e). The primer positions to amplify a region of both genes simultaneously (F7/R7) or the whole genes (\u003cem\u003eHs4\u003c/em\u003e: F5/R6; \u003cem\u003eBvHs4\u003c/em\u003e: F1/R0) are depicted by arrowheads. The figure was generated using the Illustrator for Biological Sequences software (Liu et al. 2015).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6653794/v1/7d88202f9cd20f124ff0292e.png"},{"id":84846372,"identity":"fc53c921-9ef5-4cec-bd9e-5a9db9636da9","added_by":"auto","created_at":"2025-06-18 03:08:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":166189,"visible":true,"origin":"","legend":"\u003cp\u003ePCR amplicons of \u003cem\u003eHs4\u003c/em\u003eand its \u003cem\u003eBeta\u003c/em\u003e homolog separated by agarose gel electrophoresis. Using the same set of primers, a single 1217 bp fragment was amplified in all \u003cem\u003ePatellifolia\u003c/em\u003e accessions (A), while a 1499 bp fragment was generated in most \u003cem\u003eBeta\u003c/em\u003e accessions (B). The resistant translocation lines (TR363 and TR520) and the resistant cultivar Nemata display both \u003cem\u003eHs4\u003c/em\u003e and \u003cem\u003eBvHs4\u003c/em\u003e fragments. M: 1 kb ladder; N: negative control.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6653794/v1/0fc0322ddac7d59e00021309.png"},{"id":84845678,"identity":"8231b433-4f01-4c24-a10e-977dae0471cd","added_by":"auto","created_at":"2025-06-18 03:00:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":40357,"visible":true,"origin":"","legend":"\u003cp\u003eA phylogenetic tree was created with polypeptide sequences from different \u003cem\u003ePatellifolia\u003c/em\u003e and \u003cem\u003eBeta\u003c/em\u003eaccessions. The tree was constructed using the maximum likelihood method with MEGA11 (Tamura et al. 2021). Bootstrapping was conducted with 500 replicates. Quinoa (\u003cem\u003eC. quinoa\u003c/em\u003e), spinach (\u003cem\u003eS. oleracea\u003c/em\u003e), and mung bean (\u003cem\u003eV. radiata\u003c/em\u003e) were used as outgroups.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6653794/v1/2193c81dd45e6d5f8438fb81.png"},{"id":84846823,"identity":"742a8266-86e2-484e-980f-1f7114ec286e","added_by":"auto","created_at":"2025-06-18 03:16:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":60419,"visible":true,"origin":"","legend":"\u003cp\u003eExpression levels of \u003cem\u003eHs4\u003c/em\u003e and its homologs in leaves (L) and roots (R) of resistant \u003cem\u003ePatellifolia\u003c/em\u003e(A) and susceptible \u003cem\u003eBeta\u003c/em\u003e (B) accessions as determined by RT-qPCR (primer: AK_F7/AK_R5 for \u003cem\u003eHs4\u003c/em\u003e and AS_BvHs4_F1/AS_BvHs4_R7 for \u003cem\u003eBvHs4\u003c/em\u003e; reference gene: \u003cem\u003eGAPDH\u003c/em\u003e). (A) \u003cem\u003eHs4\u003c/em\u003eexpression in fifteen \u003cem\u003ePatellifolia\u003c/em\u003e accessions and three resistant translocation lines. In all samples, the expression was higher in the roots. (B) Expression of \u003cem\u003eBvHs4\u003c/em\u003e homologs across seven different \u003cem\u003eBeta\u003c/em\u003especies. Due to a lack of biological replicates, the expression data of two different \u003cem\u003eB. macrorhiza\u003c/em\u003e accessions were pooled in this analysis. Standard deviations are given as SEM.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6653794/v1/42b2d41c26b44e60c3e5b001.png"},{"id":84845674,"identity":"49b48cda-06cc-4417-ad21-541efb258664","added_by":"auto","created_at":"2025-06-18 03:00:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":41887,"visible":true,"origin":"","legend":"\u003cp\u003eExpression levels of \u003cem\u003eHs4\u003c/em\u003e in \u003cem\u003eP. procumbens \u003c/em\u003e(primer: AK_F7/AK_R5) and \u003cem\u003eBvHs4\u003c/em\u003e in \u003cem\u003eB. vulgaris \u003c/em\u003e(primer: AS_BvHs4_F1/AS_BvHs4_R7) determined by RT-qPCR. \u003cem\u003eGAPDH\u003c/em\u003e was used as a reference. i: inoculated; ni: non-inoculated; R: root; H: hypocotyl; C: cotyledon; L: leaf. A Kruskal-Wallis test (p \u0026lt; 0.05) was performed for the \u003cem\u003eP. procumbens \u003c/em\u003esamples, and significant differences between groups were calculated using the Hochberg-corrected Dunn post-hoc test (p \u0026lt; 0.05). Statistical significances between \u003cem\u003eB. vulgaris \u003c/em\u003esamples were calculated using a one-way ANOVA, followed by the Tukey HSD Test. Standard deviations are given as SEM.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6653794/v1/ef5b60dcdfdb4f741f9d2396.png"},{"id":103765600,"identity":"c3faae58-78fc-4d6a-bfe2-fc3ac904a7ac","added_by":"auto","created_at":"2026-03-02 16:05:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1240864,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6653794/v1/5105c9ec-9590-4278-9bdb-da3b968f1681.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Subcellular localization and differential expression account for the function of the nematode resistance gene Hs4","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSugar beet, fodder beet, garden beet, and chard are diploid species (\u003cem\u003eB. vulgaris\u003c/em\u003e ssp. \u003cem\u003evulgaris, 2n\u0026thinsp;=\u0026thinsp;2x\u0026thinsp;=\u0026thinsp;18)\u003c/em\u003e belonging to the first section of the genus \u003cem\u003eBeta\u003c/em\u003e, together with the wild species \u003cem\u003eB. macrocarpa\u003c/em\u003e, \u003cem\u003eB. patula\u003c/em\u003e, \u003cem\u003eB. vulgaris\u003c/em\u003e ssp. \u003cem\u003eadanensis\u003c/em\u003e, and \u003cem\u003eB. vulgaris\u003c/em\u003e ssp. \u003cem\u003emaritima\u003c/em\u003e. The second section, \u003cem\u003eCorollinae\u003c/em\u003e, comprises the Eastern Mediterranean and Southwestern Asian species \u003cem\u003eB. corolliflora\u003c/em\u003e, \u003cem\u003eB. intermedia\u003c/em\u003e, \u003cem\u003eB. lomatogona\u003c/em\u003e, \u003cem\u003eB. macrorhiza\u003c/em\u003e, \u003cem\u003eB. nana\u003c/em\u003e, and \u003cem\u003eB. trigyna\u003c/em\u003e (Romeiras et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).The genus \u003cem\u003ePatellifolia\u003c/em\u003e was formerly described as \u003cem\u003eBeta\u003c/em\u003e section \u003cem\u003eProcumbentes\u003c/em\u003e Ulbr. but was revised to constitute a separate genus (Williams et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1976\u003c/span\u003e; Scott et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). It has diverged from the genus \u003cem\u003eBeta\u003c/em\u003e around 25.3 mya (Romeiras et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The genus forms the third gene pool and comprises the closely related diploid species \u003cem\u003eP. procumbens\u003c/em\u003e and \u003cem\u003eP. webbiana\u003c/em\u003e and the tetraploid \u003cem\u003eP. patellaris\u003c/em\u003e (Frese et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sielemann et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile cultivated beets have a narrow genetic makeup, the wild \u003cem\u003ePatellifolia\u003c/em\u003e species are genetically heterogeneous and represent an important source of resistance against diverse pathogens like the Beet Necrotic Yellow Vein Virus (vectored by the fungus \u003cem\u003ePolymyxa betae)\u003c/em\u003e and the beet cyst nematode (BCN) \u003cem\u003eHeterodera schachtii\u003c/em\u003e (Panella et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, the different species were employed extensively in crossing experiments with various \u003cem\u003eBeta\u003c/em\u003e species, but the lack of chromosome homology and pronounced hybrid sterility severely hindered their use as genetic resources.\u003c/p\u003e \u003cp\u003eSugar beet is highly susceptible to BCN, which attacks the roots and induces a nutrient cell called syncytium (Sijmons et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Wyss \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). After passing through four larval stages, the maturing female forms a cyst filled with eggs. The syncytium is the only feeding source for the nematodes, and its development depends on its integrity. The BCN is the most significant pest in beet cultivation (M\u0026uuml;ller \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Daub \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). There is no complete resistance within the \u003cem\u003eBeta\u003c/em\u003e genus. In contrast, the \u003cem\u003ePatellifolia\u003c/em\u003e species are entirely resistant to the pathogen. However, transferring the resistance genes is extremely difficult due to crossing barriers. After decades of research, only a few resistant beet lines have been selected carrying translocations from \u003cem\u003eP. procumbens\u003c/em\u003e and \u003cem\u003eP. webbiana\u003c/em\u003e (Savitsky \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; Jung and Wricke \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1987\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecently, \u003cem\u003eHs4\u003c/em\u003e was cloned from a \u003cem\u003ePatellifolia\u003c/em\u003e translocation attached to beet chromosome 9 (Kumar et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Knocking out \u003cem\u003eHs4\u003c/em\u003e turned the resistant sugar beet variety Nemata into a highly susceptible one. In contrast, \u003cem\u003eHs4\u003c/em\u003e overexpression in a susceptible \u003cem\u003eB. vulgaris\u003c/em\u003e background resulted in varying resistance depending on the gene expression level. Hs4 was predicted to be an Endoplasmic Reticulum (ER)-bound rhomboid-like protease of 210 amino acids. Most plant rhomboid proteases contain seven transmembrane domains (TMDs). They are characterized by a serine protease catalytic unit that cleaves the substrates inside the membrane. Rhomboid proteins can also lack the catalytic residues, rendering them inactive proteases, and are then classified as iRhoms (Freeman \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Urban and Dickey \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). A \u003cem\u003eB. vulgaris\u003c/em\u003e homolog (BvHs4) showed 60 % polypeptide similarity to Hs4. It differs from Hs4 by102 additional amino acids at the N-terminus, and it lacks the leader sequence that directs Hs4 to the ER. Therefore, differences in subcellular localization of the two homologs are highly likely.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to determine whether functional orthologs of \u003cem\u003eHs4\u003c/em\u003e exist in other \u003cem\u003ePatellifolia\u003c/em\u003e and \u003cem\u003eBeta\u003c/em\u003e species. We reasoned that the highly conserved structure of the Hs4-like proteins points to their function as BCN resistance genes in other species. We found highly similar \u003cem\u003eHs4\u003c/em\u003e orthologs in all \u003cem\u003ePatellifolia\u003c/em\u003e species, whereas \u003cem\u003eBvHs4\u003c/em\u003e-homologous genes in \u003cem\u003eBeta\u003c/em\u003e species were highly divergent. Because all \u003cem\u003eBeta\u003c/em\u003e species are susceptible, we reasoned that the lack of function could be due to the ectopic expression. Therefore, we determined the local expression of \u003cem\u003eHs4\u003c/em\u003e homologs in a broad set of species in different tissues and under nematode infestation. We found that \u003cem\u003eHs4\u003c/em\u003e was highest expressed in roots while \u003cem\u003eBvHs4\u003c/em\u003e expression peaked in leaves. The results suggest that \u003cem\u003eHs4\u003c/em\u003e and its \u003cem\u003ePatellifolia\u003c/em\u003e orthologs are the only functional nematode-resistance genes. These results shed new light on the function of \u003cem\u003eHs4\u003c/em\u003e and have implications for breeding nematode-resistant \u003cem\u003eBeta\u003c/em\u003e crops.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eSequence variations between \u003cem\u003eHs4\u003c/em\u003e and its homologs from \u003cem\u003ePatellifolia\u003c/em\u003e and \u003cem\u003eBeta\u003c/em\u003e species\u003c/p\u003e \u003cp\u003eThe published \u003cem\u003eHs4\u003c/em\u003e sequence comprises 5664 bp with five exons and five introns (Kumar et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This sequence contained a large stretch (964 bp) of unknown nucleotides in the first intron. We sequenced the unknown region to 107 bp, thereby reducing the genomic region of \u003cem\u003eHs4\u003c/em\u003e to 4999 bp (Supplementary Fig.\u0026nbsp;1). The composition of the cDNA and the resulting protein remained unchanged. In the following, '\u003cem\u003eHs4'\u003c/em\u003e refers to the 4999 bp long sequence.\u003c/p\u003e \u003cp\u003eUsing the primer combination AS_F7/AS_R7, we detected \u003cem\u003eHs4\u003c/em\u003e amplicons in all \u003cem\u003eP. procumbens\u003c/em\u003e, \u003cem\u003eP. webbiana\u003c/em\u003e, and \u003cem\u003eP. patellaris\u003c/em\u003e accessions analyzed. The \u003cem\u003eP. procumbens\u003c/em\u003e, \u003cem\u003eP. webbiana\u003c/em\u003e, and \u003cem\u003eP. patellaris\u003c/em\u003e homologs were named \u003cem\u003ePpHs4, PwHs4\u003c/em\u003e, and \u003cem\u003ePpatHs4\u003c/em\u003e, respectively. Additionally, we screened the sugar beet translocation lines TR363 and TR520 and the hybrid variety Nemata with the same primer set. The primers also bind to the exons of the \u003cem\u003eB. vulgaris Hs4\u003c/em\u003e homolog (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), so double bands could be observed after agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The lower and upper bands are \u003cem\u003eP. procumbens\u003c/em\u003e (\u003cem\u003eHs4\u003c/em\u003e, 1217 bp) and \u003cem\u003eB. vulgaris\u003c/em\u003e (\u003cem\u003eBvHs4\u003c/em\u003e, 1499 bp) amplicons.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe expected genes identified from \u003cem\u003eP. procumbens\u003c/em\u003e to exhibit the closest sequence similarity to \u003cem\u003eHs4\u003c/em\u003e. Due to the close relatedness between \u003cem\u003eP. procumbens\u003c/em\u003e and \u003cem\u003eP. webbiana\u003c/em\u003e, we expected a high similarity between \u003cem\u003ePpHs4\u003c/em\u003e and \u003cem\u003ePwHs4\u003c/em\u003e sequences, while \u003cem\u003ePpatHs4\u003c/em\u003e should display less similarity to \u003cem\u003eHs4\u003c/em\u003e. As a first result, \u003cem\u003eHs4\u003c/em\u003e was conserved across all \u003cem\u003ePatellifolia\u003c/em\u003e species. Apart from single nucleotide polymorphisms (SNPs) and short insertions/deletions (InDels) (Supplementary Table\u0026nbsp;3), the protein-coding exons 2\u0026ndash;6 (Supplementary Fig.\u0026nbsp;3A) were highly conserved. The overall sequence identity was 95\u0026ndash;99% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As expected, the \u003cem\u003eHs4\u003c/em\u003e sequences from the translocation lines (\u003cem\u003eHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eTR363/TR520\u003c/em\u003e\u003c/sub\u003e) and Nemata (\u003cem\u003eHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eNemata\u003c/em\u003e\u003c/sub\u003e) were 100% identical to \u003cem\u003eHs4\u003c/em\u003e, thus confirming their origin. Moreover, \u003cem\u003ePwHs4\u003c/em\u003e sequences showed the highest identity to \u003cem\u003eHs4\u003c/em\u003e (99%). Identity values between \u003cem\u003eHs4\u003c/em\u003e on one side and \u003cem\u003ePpHs4\u003c/em\u003e and \u003cem\u003ePpatHs4\u003c/em\u003e were lower (95\u0026ndash;99%) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These sequences exhibited InDels extending the sequence by 29\u0026ndash;30 bp (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Accordingly, most SNPs were shared between \u003cem\u003eP. procumbens\u003c/em\u003e and \u003cem\u003eP. patellaris\u003c/em\u003e accessions. Four InDels were located within intronic regions. Moreover, a three bp insertion (position 1302, exon 5) led to the addition of one amino acid (Supplementary Fig.\u0026nbsp;4, Supplementary Fig.\u0026nbsp;8, Supplementary Table\u0026nbsp;3). This insertion was present in four \u003cem\u003eP. procumbens\u003c/em\u003e, two \u003cem\u003eP. webbiana\u003c/em\u003e, and all \u003cem\u003eP. patellaris\u003c/em\u003e accessions, as well as in the \u003cem\u003eP. procumbens\u003c/em\u003e draft genome sequence.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDNA sequence identities of different \u003cem\u003eHs4\u003c/em\u003e sequences compared to the \u003cem\u003eHs4\u003c/em\u003e reference ORF of 2022 bp. The \u003cem\u003eHs4\u003c/em\u003e homologs' suffix denotes the seed code or other identifiers. Similarities were calculated with the blastn suite (Camacho et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuery sequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLength\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIdentity [%]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGaps [%]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHs4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePpHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003e100810\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2051\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePpHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003e100820\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2052\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePpHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003e100821\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePpHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eIPK419\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePpHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eIPK951\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePwHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003e100002\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePwHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003e100832\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePwHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003e100833\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePwHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eIPK526\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePwHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eIPK927\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePpatHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003e100012\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2052\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePpatHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003e930065\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2052\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePpatHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eIPK10\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2052\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePpHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eDraft\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eTR363\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eTR520\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eNemata\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eBvHs4\u003c/em\u003e homologs are present in a broad set of \u003cem\u003eBeta\u003c/em\u003e species.\u003c/p\u003e \u003cp\u003eBased on the phylogeny of the genus \u003cem\u003eBeta\u003c/em\u003e, we expected \u003cem\u003eBvHs4\u003c/em\u003e-like sequences in species of the genus \u003cem\u003eBeta\u003c/em\u003e. We used the primer combination AS_F7/AS_R7 amplifying 1499 bp of \u003cem\u003eBvHs4\u003c/em\u003e to screen 14 \u003cem\u003eBeta\u003c/em\u003e accessions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). As a result, amplicons of the expected size were visible in 12 accessions. The \u003cem\u003eB. macrocarpa\u003c/em\u003e and \u003cem\u003eB. patula\u003c/em\u003e amplicons were substantially shorter (\u003cem\u003eBmrHs4\u003c/em\u003e, ca. 1300 bp; \u003cem\u003eBpHs4\u003c/em\u003e, ca. 1300 bp). \u003cem\u003eB. corolliflora\u003c/em\u003e (\u003cem\u003eBcHs4\u003c/em\u003e) exhibited an additional amplicon ca. 1300 bp in size (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Sequencing the smaller \u003cem\u003eB. corolliflora\u003c/em\u003e fragment revealed no homology to any known gene from \u003cem\u003eB. vulgaris\u003c/em\u003e (data not shown), while the larger \u003cem\u003eBcHs4\u003c/em\u003e fragment was composed of different sequences. \u003cem\u003eBcHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e was a perfect match to \u003cem\u003eBvHs4\u003c/em\u003e from the reference genome sequence, while another sequence (\u003cem\u003eBcHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eB\u003c/em\u003e\u003c/sub\u003e) showed several SNPs, small InDels, and a prominent insertion of 97 bp (after 1522 bp of the \u003cem\u003eBvHs4\u003c/em\u003e RefBeet sequence) matching a \u003cem\u003eBvHs4\u003c/em\u003e homolog from the tolerant \u003cem\u003eB. vulgaris\u003c/em\u003e genotype U2Bv. \u003cem\u003eBcHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eB\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eBvHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eU2Bv\u003c/em\u003e\u003c/sub\u003e displayed a 312 bp deletion (after 2713 bp of \u003cem\u003eBvHs4\u003c/em\u003e) (Supplementary Fig.\u0026nbsp;5B). We then used a \u003cem\u003eBvHs4\u003c/em\u003e-specific primer combination (AS_BvHs4_F1/AS_BvHs4_R0, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) for amplifying the whole \u003cem\u003eBvHs4\u003c/em\u003e open reading frame of 4342 bp from seven \u003cem\u003eBeta\u003c/em\u003e accessions (Supplementary Fig.\u0026nbsp;6). Here, we found the 97 bp insertion and the 312 bp deletion to also be present in \u003cem\u003eBpHs4\u003c/em\u003e and \u003cem\u003eBmrHs4\u003c/em\u003e. Further, they contained the same small InDels found in \u003cem\u003eBcHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eB\u003c/em\u003e\u003c/sub\u003e. \u003cem\u003eBpHs4, BcHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eB\u003c/em\u003e\u003c/sub\u003e, and \u003cem\u003eBvHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eU2Bv\u003c/em\u003e\u003c/sub\u003e shared single SNPs but not \u003cem\u003eBmrHs4\u003c/em\u003e (Supplementary Fig.\u0026nbsp;4). Excluding \u003cem\u003eBmrHs4\u003c/em\u003e, which shows exonic deviations of up to 15 bp, all sequence variations larger than three basepairs lie within intronic regions (Supplementary Fig.\u0026nbsp;3B, Supplementary Fig.\u0026nbsp;5B).\u003c/p\u003e \u003cp\u003eIn summary, the \u003cem\u003eBvHs4\u003c/em\u003e homologs from species of the genus \u003cem\u003eBeta\u003c/em\u003e are highly similar (sequence identity values 97\u0026ndash;100%), and the intron/exon structures are the same as in \u003cem\u003eBvHs4\u003c/em\u003e (Supplementary Fig.\u0026nbsp;3). None of the genes had higher similarities to \u003cem\u003eHs4\u003c/em\u003e than \u003cem\u003eBvHs4\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In conclusion, none of the \u003cem\u003eBeta Hs4\u003c/em\u003e homologs will likely function as a nematode resistance gene.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDNA sequence identities of different \u003cem\u003eBvHs4\u003c/em\u003e homologs compared to the \u003cem\u003eBvHs4\u003c/em\u003e (4342 bp) and \u003cem\u003eHs4\u003c/em\u003e (2022 bp) ORFs. The \u003cem\u003eHs4\u003c/em\u003e homologs' suffix denotes the seed code or other identifiers. Similarities were calculated with the blastn suite (Camacho et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003ecompared to \u003cem\u003eBvHs4\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003ecompared to \u003cem\u003eHs4\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuery sequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLength\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIdentity [%]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGaps [%]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIdentity [%]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGaps [%]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBaHs4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4341\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBcHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4343\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBcHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eB\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBmHs4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4347\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBmHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eBmar1.0\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4369\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBmcHs4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4332\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBmrHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003e960018\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4166\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBpHs4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4166\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBpHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eBpat-1.0\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBvHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eEL10_2\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4334\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBvHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eW357B\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4342\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBvHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eU2Bv\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHs4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePhylogenetic analysis indicates distinct clades of Hs4- and BvHs4-like polypeptides\u003c/p\u003e \u003cp\u003eWe compared the open reading frames of all \u003cem\u003eBeta\u003c/em\u003e and \u003cem\u003ePatellifolia\u003c/em\u003e accessions together with the rhomboid-like proteins of quinoa (\u003cem\u003eChenopodium quinoa\u003c/em\u003e, LOC110702170 and LOC110705623), mung bean (\u003cem\u003eVigna radiata\u003c/em\u003e, LOC106763922), and spinach (\u003cem\u003eSpinacia oleracea\u003c/em\u003e, LOC110775063) as outgroups.\u003c/p\u003e \u003cp\u003eApart from minor amino acid substitutions, the polypeptides from the \u003cem\u003ePatellifolia\u003c/em\u003e species showed high conservation (Supplementary Fig.\u0026nbsp;8). A single amino acid insertion (after Hs4 position 132) compared to Hs4 was present in various accessions of all three \u003cem\u003ePatellifolia\u003c/em\u003e species (Supplementary Fig.\u0026nbsp;7).\u003c/p\u003e \u003cp\u003eThe sequence motif 'LLRDRCPDN' was suggested to be Hs4-specific due to its absence from BvHs4 (Kumar et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Surprisingly, it could only be found in four out of thirteen \u003cem\u003ePatellifolia\u003c/em\u003e sequences, the translocation lines, and Nemata (Supplementary Table\u0026nbsp;4). Because all \u003cem\u003ePatellifolia\u003c/em\u003e polypeptides carried two more amino acids at their 3\u0026rsquo;-end, which were absent from the \u003cem\u003eBeta\u003c/em\u003e polypeptides, the conserved motif could be revised to 'CPDNKE'.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eBeta\u003c/em\u003e polypeptides were highly similar to each other and to the outgroup proteins (Supplementary Fig.\u0026nbsp;8). The minor sequence variations of the BmrHs4 sequence may result in a non-functional polypeptide because of a stop codon at position 141 (Supplementary Fig.\u0026nbsp;8).\u003c/p\u003e \u003cp\u003eAcross the two genera, motives of up to fifteen amino acids (aa) were conserved. A prominent difference was the addition of roughly 100 aa at the N-terminus of all \u003cem\u003eBeta\u003c/em\u003e proteins, resulting from an additional exon (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThen, we generated a maximum likelihood tree based on the polypeptide sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The BvHs4 homologs clustered closest to the outgroup proteins, and the \u003cem\u003ePatellifolia\u003c/em\u003e polypeptides were grouped further away. The \u003cem\u003eBeta\u003c/em\u003e species formed a distinct clade, except for \u003cem\u003eB. macrorhiza\u003c/em\u003e. The divergence point towards the \u003cem\u003ePatellifolia\u003c/em\u003e polypeptides was supported by a high bootstrap value of 100, indicating that this clade is distinct from the \u003cem\u003eBeta\u003c/em\u003e and the outgroup proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Conclusively, the original Hs4 polypeptide grouped with the translocation lines and Nemata, which were all derived from the same \u003cem\u003eP. procumbens\u003c/em\u003e source. Additionally, one PpHs4 and three PwHs4 polypeptides are part of that subclade. Interestingly, onePpHs4 and the remaining PwHs4 polypeptides formed a distinct subclade. The polypeptide derived from the \u003cem\u003eP. procumbens\u003c/em\u003e draft genome was somewhat distantly related to Hs4. Unexpectedly, three \u003cem\u003eP. procumbens\u003c/em\u003e accessions grouped with the \u003cem\u003eP. patellaris\u003c/em\u003e accessions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProtein localization predictions underpin the diversification of Hs4 homologs across species of the genus \u003cem\u003eBeta\u003c/em\u003e\u003c/p\u003e \u003cp\u003eHs4 was predicted to be localized within the ER membrane (Kumar et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). We analyzed the protein structure with DeepTMHMM (Hallgren et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), which predicts that Hs4 has six transmembrane domains (Supplementary Table\u0026nbsp;5). The added valine in PpHs4\u003csub\u003e100810\u003c/sub\u003e did not change its subcellular localization. Contrarily, the probability of an ER localization increased from 0.7973 for Hs4 to 0.8184 for PpHs4\u003csub\u003e100810\u003c/sub\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, the additional valine led to a slight conformation change. Five amino acids assigned to a transmembrane domain in Hs4 were now predicted to be part of a loop located in the lumen, and hence, the proximate fourth transmembrane domain was slightly decreased in size (Supplementary Table\u0026nbsp;5).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePredicted localization and membrane association for Hs4 and its homologs from \u003cem\u003ePatellifolia\u003c/em\u003e and \u003cem\u003eBeta\u003c/em\u003e species compared to the \u003cem\u003eA. thaliana\u003c/em\u003e rhomboid protease AtRBL11. The probability values were calculated using DeepLoc2.1 (\u0026Oslash;dum et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHs4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePpHs4\u003csub\u003e100810\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBvHs4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAtRBL11\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLocalization\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003ePrediction probability\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0667\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0695\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0707\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0796\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003enucleus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0761\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0790\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0566\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0689\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eextracellular\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1902\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1712\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0209\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecell membrane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1334\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1330\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0505\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0623\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emitochondrion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.3080\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.2782\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2748\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.3320\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eplastid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1721\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.2045\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.9346\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eendoplasmic reticulum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.7973\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.8184\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2247\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1656\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003elysosome/vacuole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.4065\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4335\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0581\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0521\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGolgi apparatus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.4089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4283\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0673\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0372\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eperoxisome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0371\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1345\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMembrane association\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eperipheral\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1530\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2850\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.3000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etransmembrane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.9670\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9680\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.9610\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003elipid anchor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0740\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0770\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0590\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0550\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esoluble\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1430\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1450\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBvHs4, in turn, is predicted to be a plastid-localized transmembrane protein exhibiting six transmembrane domains (prediction value 0.9158) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The main conformation difference lies in its long cytosolic N-stretch, which is much shorter in Hs4 and PpHs4\u003csub\u003e100810\u003c/sub\u003e (Supplementary Table\u0026nbsp;5). Interestingly, using the AlphaFold Protein Structure Database (Varadi et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Varadi et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), we found that Arabidopsis contains an Hs4 homolog with 57 % and 71 % polypeptide similarity to Hs and BvH4, respectively. The protein is classified as a rhomboid-like protease 11 (AtRBL11, UniProt identifier: Q84MB5) and, like BvHs4, located in the plastids (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). It also has six transmembrane domains (Supplementary Table\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003cem\u003eHs4\u003c/em\u003e and its homologs are differentially regulated between \u003cem\u003ePatellifolia\u003c/em\u003e and \u003cem\u003eBeta\u003c/em\u003e species\u003c/p\u003e \u003cp\u003eWe measured the transcriptional activities of \u003cem\u003eHs4\u003c/em\u003e and its homologs in \u003cem\u003ePatellifolia\u003c/em\u003e and \u003cem\u003eBeta\u003c/em\u003e species with and without nematode infection. The translocation lines and the \u003cem\u003ePatellifolia\u003c/em\u003e species displayed up to 26.6-fold higher \u003cem\u003eHs4\u003c/em\u003e expression in roots than in leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Contrastingly, across all \u003cem\u003eBeta\u003c/em\u003e species, the \u003cem\u003eBvHs4\u003c/em\u003e homologs showed up to 6x higher expression in leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The leaf-to-root expression ratio was similar across the different species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we analyzed the expression with and without nematode infection. After infection with \u003cem\u003eH. schachtii\u003c/em\u003e, no females or cysts were observed on \u003cem\u003eP. procumbens\u003c/em\u003e roots, whereas \u003cem\u003eB. vulgaris\u003c/em\u003e roots were heavily infected. Female numbers ranged between 54 and 119 (mean: 83.4). \u003cem\u003eP. procumbens\u003c/em\u003e showed the highest \u003cem\u003eHs4\u003c/em\u003e expression in roots across all sampling points (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The root/leaf ratio did not differ much between non-inoculated and inoculated plants. Upregulation after nematode inoculation was not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In the young sugar beet plants, \u003cem\u003eBvHs4\u003c/em\u003e expression was higher in roots than in leaves. However, shortly before inoculation, \u003cem\u003eBvHs4\u003c/em\u003e expression in leaves was 4.1x higher than in roots. The \u003cem\u003eBvHs4\u003c/em\u003e expression rates did not respond to nematode infection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we revised the genomic sequence of the nematode resistance gene \u003cem\u003eHs4\u003c/em\u003e (Kumar et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)We studied Hs4 homologs in various Patellifolia and Beta species and examined their expression in different tissues with and without \u003cem\u003eH. schachtii\u003c/em\u003e infestations.\u003c/p\u003e \u003cp\u003e \u003cem\u003eHs4\u003c/em\u003e has been described to span 5664 bp (Kumar et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). After sequencing an unresolved region of 964 bp in the first intron, we found that \u003cem\u003eHs4\u003c/em\u003e comprises 4999 bp. Kumar et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) claimed the gene to consist of five exons and introns each, disregarding the untranslated regions (UTRs) they had annotated (see Supplementary Fig.\u0026nbsp;1). Exons do not necessarily have to be protein-coding. Instead, UTRs are also considered exonic regions, though non-coding (Aspden et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Hence, according to our studies, \u003cem\u003eHs4\u003c/em\u003e consists of six exons and five introns.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eHs4\u003c/em\u003e gene was predicted to encode a rhomboid protease (Kumar et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). According to Urban and Dickey (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), the smallest catalytically active unit of rhomboid proteases consists of six transmembrane domains (TMD). These basic TMDs are mainly found in bacteria and partly in eukaryotes, though eukaryotes often have a seventh TMD (Urban and Dickey \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). However, many eukaryotic rhomboids belong to the secretase subfamily, which can be divided into two clades: secretase-A rhomboids exhibit seven TMDs, and secretase-B rhomboids contain only the core region of six TMDs. Further, these two clades are differentiated by distinct sequences around the catalytic serine S106. In the secretase-A clade, proteins contain a highly conserved GxSxGVYA, while B-class secretases show a less stringent GxSxxxF sequence (Lemberg and Freeman \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). When we analyzed Hs4 using DeepTMHMM, we found the protein to consist of six TMDs, which is conclusive in its classification as a rhomboid protease. Further, Hs4 (and all related proteins analyzed in the current study) harbors the GxSxxxF sequence, indicating its affiliation with the secretase-B clade.\u003c/p\u003e \u003cp\u003eSpecies of the genus \u003cem\u003ePatellifolia\u003c/em\u003e are highly resistant to \u003cem\u003eH. schachtii\u003c/em\u003e (Golden \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1959\u003c/span\u003e; Viglierchio \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1960\u003c/span\u003e). According to our hypothesis, we detected highly similar homologs in all \u003cem\u003ePatellifolia\u003c/em\u003e species. Nevertheless, sequence variations could be observed between and within the species. Sequences perfectly matching \u003cem\u003eHs4\u003c/em\u003e were detected in the translocation lines and the hybrid variety Nemata, reassuring the origin of the \u003cem\u003eHs4\u003c/em\u003e sequence. None of the \u003cem\u003ePatellifolia\u003c/em\u003e accessions sequenced harbored a homolog with 100 % sequence identity to \u003cem\u003eHs4\u003c/em\u003e. Hoever, \u003cem\u003ePpHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eIPK419\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003ePpHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eIPK951\u003c/em\u003e\u003c/sub\u003e, and all \u003cem\u003ePwHs4\u003c/em\u003e homologs exhibited the highest similarity to \u003cem\u003eHs4\u003c/em\u003e (99 %). Three base pairs were interated into exon 5, adding valine to the Hs4 protein present in all three species. However, we found that the additional amino acid changed neither the localization of Hs4 to the ER nor its TMD structure.\u003c/p\u003e \u003cp\u003eThe phylogenetic relationships among the different \u003cem\u003ePatellifolia\u003c/em\u003e accessions have long since been discussed. While the diploid \u003cem\u003eP. procumbens\u003c/em\u003e and \u003cem\u003eP. webbiana\u003c/em\u003e were previously considered two extreme ecotypes of a single species (Curtis \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1968\u003c/span\u003e; Wagner et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), recent phylogenetic analyses based on plastome data have pointed to the clear separation into two distinct albeit closely related species. The tetraploid \u003cem\u003eP. patellaris\u003c/em\u003e is more distantly related to the two diploid \u003cem\u003ePatellifolia\u003c/em\u003e species (Sielemann et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In our study, two \u003cem\u003ePpHs4\u003c/em\u003e and all \u003cem\u003ePwHs4\u003c/em\u003e homologs showed high sequence similarity to \u003cem\u003eHs4\u003c/em\u003e. Nevertheless, three \u003cem\u003ePpHs4\u003c/em\u003e and the \u003cem\u003ePpatHs4\u003c/em\u003e homologs showed several deviations from \u003cem\u003eHs4\u003c/em\u003e. Considering the relationship between the \u003cem\u003ePatellifolia\u003c/em\u003e species, similarities between \u003cem\u003eP. procumbens\u003c/em\u003e and \u003cem\u003eP. patellaris\u003c/em\u003e appear peculiar. However, though \u003cem\u003eP. procumbens\u003c/em\u003e and \u003cem\u003eP. webbiana\u003c/em\u003e are more closely related, the genus itself is very diverse. Depending on sampling locations, plant morphologies differ even within species (Frese et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This was underpinned by molecular marker studies demonstrating high genetic diversity within \u003cem\u003eP. procumbens\u003c/em\u003e (Nachtigall et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSequence variations between \u003cem\u003eHs4\u003c/em\u003e homologs from different \u003cem\u003ePatellifolia\u003c/em\u003e species have been recently described (Reeves and Richards \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Analyzing short haplotype loci polymorphic between different species, they identified 0.16\u0026ndash;3.36 variants between \u003cem\u003eHs4\u003c/em\u003e orthologs. Of these loci, 20 % contained single- or muti-nucleotide polymorphisms, and the remaining 80 % PAVs. Strikingly, Reeve and Richards (2022) detected differences in exonic variants at several \u003cem\u003eHs4\u003c/em\u003e loci across accessions within the same species, which aligns with our results. In addition, we identified a major InDel in exon five of several \u003cem\u003ePatellifolia\u003c/em\u003e accessions, resulting in an added valine, which had not been reported in the study by Reeves and Richards (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Instead, they reported four short haplotype loci with differing major variants in or around exon five, which, in turn, could not be detected in our study.\u003c/p\u003e \u003cp\u003eDue to the high similarities between \u003cem\u003eHs4\u003c/em\u003e and \u003cem\u003ePwHs4\u003c/em\u003es from \u003cem\u003eP. webbiana\u003c/em\u003e, we speculate that \u003cem\u003eHs4\u003c/em\u003e might originate from \u003cem\u003eP. webbiana\u003c/em\u003e. A second resistance gene is on chromosome 7 of \u003cem\u003eP. procumbens\u003c/em\u003e and \u003cem\u003eP. webbiana\u003c/em\u003e (Jung et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). Using the primers matching \u003cem\u003eHs4\u003c/em\u003e, no other \u003cem\u003eHs4\u003c/em\u003e homologous sequence could be amplified from the \u003cem\u003ePatellifolia\u003c/em\u003e species, suggesting no \u003cem\u003eHs4\u003c/em\u003e paralog on chromosome 7.\u003c/p\u003e \u003cp\u003eThe genera \u003cem\u003ePatellifolia\u003c/em\u003e and \u003cem\u003eBeta\u003c/em\u003e have diverged 25 mya (Romeiras et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The general sequence similarity between sugar beet and \u003cem\u003ePatellifolia\u003c/em\u003e genomes is 75 % (Reeves and Richards \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Apart from \u003cem\u003eBvHs4\u003c/em\u003e,we detected a highly similar homolog in the wild progenitor of sugar beet, \u003cem\u003eB. vulgaris\u003c/em\u003e ssp. \u003cem\u003emaritima\u003c/em\u003e. As expected, the tetraploid species \u003cem\u003eB. corolliflora\u003c/em\u003e (Sielemann et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e) has two \u003cem\u003eBvHs4\u003c/em\u003e-homologous sequences that varied at several sites. While \u003cem\u003eBcHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e resembled \u003cem\u003eBvHs4\u003c/em\u003e, \u003cem\u003eBcHs4\u003c/em\u003e\u003csub\u003e\u003cem\u003eB\u003c/em\u003e\u003c/sub\u003e had a prominent insertion of 97 bp and a deletion of 312 bp, both of which were also present in \u003cem\u003eB. patula\u003c/em\u003e and \u003cem\u003eB. macrorhiza\u003c/em\u003e. Matching these results, \u003cem\u003eB. macrorhiza\u003c/em\u003e is believed to be a parental species of \u003cem\u003eB. corolliflora\u003c/em\u003e (Sielemann et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). The insertion is similar to the \u003cem\u003eBvHs4\u003c/em\u003e homolog from the nematode-tolerant sugar beet genotype U2Bv. This genotype is characterized as nematode tolerant based on the expression of two \u003cem\u003eBvNLP7\u003c/em\u003e genes that are located on chromosome 5 (Sielemann et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e) and which do not show any resemblance to the nematode resistance gene \u003cem\u003eHs4\u003c/em\u003e, whose \u003cem\u003eBeta\u003c/em\u003e homolog is located on chromosome 2. Both \u003cem\u003eBcHs4\u003c/em\u003e variants lie in the intron, so they do not affect the resulting protein. Further, these sequences did not show higher similarity to \u003cem\u003eHs4\u003c/em\u003e than \u003cem\u003eBvHs4\u003c/em\u003e and are not expected to contribute to nematode resistance.\u003c/p\u003e \u003cp\u003eWe were interested in the expression patterns of the \u003cem\u003eHs4\u003c/em\u003e homologs from other species. \u003cem\u003eHs4\u003c/em\u003e is highly expressed in roots and less in leaves. Noteworthy, we detected the highest expression levels in the translocation line TR520 and the hybrid variety Nemata derived from it. A second translocation line, TR363, did not show enhanced \u003cem\u003eHs4\u003c/em\u003e expression levels. TR363 harbors a much smaller translocation than TR520 (Kumar et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and important regulatory \u003cem\u003ecis\u003c/em\u003e-elements might be lacking in its sequence, leading to differences in expression intensities. TR363 never gained importance in beet breeding due to its inferior yield and quality characteristics.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBvHs4\u003c/em\u003e, on the other hand, is expressed strongly in leaves. Its protein is predicted to target the chloroplast, similar to its \u003cem\u003eArabidopsis\u003c/em\u003e homolog AtRBL11 (Kmiec-Wisniewska et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Its strikingly different expression pattern and low sequence homology to \u003cem\u003eHs4\u003c/em\u003e indicate that \u003cem\u003eBvHs4\u003c/em\u003e and its homologs from other \u003cem\u003eBeta\u003c/em\u003e species do not function as nematode-resistance genes. We speculate that \u003cem\u003eHs4\u003c/em\u003e has acquired a new function in evolution, coupled with its localization in the ER membrane, which provides a typical example of the neo-functionalization of a gene. In conclusion, the significant structural differences between Hs4 and its Beta homologs and their different expression patterns prohibit a targeted modification of their function, e.g., by genome editing to convert them into resistance genes. Due to the poor agronomic performance of the nematode-resistant translocation lines, the expression of the \u003cem\u003eHs4\u003c/em\u003e gene after transformation into sugar beet is the only realistic solution to breed resistant varieties.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003ePlant material and growth conditions\u003c/p\u003e \u003cp\u003eFifteen \u003cem\u003eBeta\u003c/em\u003e and fifteen \u003cem\u003ePatellifolia\u003c/em\u003e accessions, the resistant translocation lines TR363 and TR520, and the resistant variety Nemata derived from TR520 (Supplementary Table\u0026nbsp;1) were grown in a greenhouse or a climate chamber, respectively, under long-day conditions (LD; 16 h light / 8 h dark).\u003c/p\u003e \u003cp\u003eDNA isolation and PCR\u003c/p\u003e \u003cp\u003eSamples of different plant tissues were taken at several points, frozen in liquid nitrogen, and stored at -70\u0026deg;C until further usage. Following the grinding of the tissues, genomic DNA was isolated using the NucleoSpin Plant II kit (Macherey-Nagel, D\u0026uuml;ren, Germany) following the manufacturer's instructions or the plant DNA mini preparation protocol (Dellaporta et al. 1983). Before further usage, the genomic DNA was checked on a 1% agarose gel (90V, 30 min or 80V, 12 min).\u003c/p\u003e \u003cp\u003eA non-sequenced (unresolved) region in the first \u003cem\u003eHs4\u003c/em\u003e intron was amplified from the variety Nemata (Supplementary Table\u0026nbsp;1) with a Taq polymerase (Biozym Scientific GmbH, Hessisch Oldendorf, Germany) using the primer combination AK_F2/AS_R5 (Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eTo amplify putative homologs of \u003cem\u003eHs4\u003c/em\u003e and \u003cem\u003eBvHs4\u003c/em\u003e simultaneously, we designed a primer set (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) binding to a DNA region encoding a highly conserved polypeptide sequence between Hs4 and BvHs4 (Supplementary Fig.\u0026nbsp;2). The primer pair AS_F7/AS_R7 was expected to amplify 1217 bp and 1499 bp of the \u003cem\u003eHs4\u003c/em\u003e and \u003cem\u003eBvHs4\u003c/em\u003e genes (Supplementary Table\u0026nbsp;2). Taq polymerase (Biozym Scientific GmbH) or Phusion\u0026trade; High-Fidelity polymerase (ThermoFisher Scientific\u0026trade;, Waltham, MA, USA) was used for amplifying the genomic DNA of \u003cem\u003eHs4\u003c/em\u003e and its homologs from other species. The primer combination AK_F5/AK_R6 (Supplementary Table\u0026nbsp;2) was used for \u003cem\u003eHs4\u003c/em\u003e ORF amplification whereas AS_BvHs4_F1/AS_BvHs4_R0 (Supplementary Table\u0026nbsp;2) was used to amplify the Beta homolog ORFs. The PCR products were analyzed on a 1% agarose gel (90V, 30\u0026ndash;40 min).\u003c/p\u003e \u003cp\u003ePlasmid cloning and Sanger sequencing\u003c/p\u003e \u003cp\u003eUsing the CloneJET PCR Cloning Kit (ThermoFisher Scientific\u0026trade;), DNA was cloned into the pJET1.2/blunt cloning vector according to the manufacturer's instructions and transformed into competent \u003cem\u003eEscherichia coli\u003c/em\u003e (strain DH5α; DNA Cloning Service eK, Hamburg, Germany) via the heat-shock method (Froger and Hall \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Following overnight incubation at 37\u0026deg;C on LB plates supplemented with ampicillin (50 \u0026micro;g/ml), colonies were screened for the plasmid's presence by PCR. Positive colonies were cultured overnight in 5 ml LB with ampicillin on a shaker. Plasmids were then isolated using the NucleoSpin Plasmid QuickPure Kit (Macherey-Nagel) and used for PCR in a 1:1000 dilution.\u003c/p\u003e \u003cp\u003eAmplified gene fragments or whole genes were Sanger sequenced on an Applied Biosystems 3730xl DNA Analyzer (ThermoScientific\u0026trade;, via IKMB, Kiel University) using a set of different primers (Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003ePhylogenetic analysis\u003c/p\u003e \u003cp\u003eSanger sequences were aligned to the reference sequences using the CLC Main Workbench 20 (Qiagen, Hilden, Germany). As a reference, the \u003cem\u003eHs4\u003c/em\u003e gene sequence derived from TR520 by Kumar et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) was used for the \u003cem\u003ePatellifolia\u003c/em\u003e sequences, and the genomic RefBeet-1.2.2 (Dohm et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) sequence of the rhomboid-like protein 11 from sugar beet (NCBI Reference Sequence XM_010669575.1) was deployed for all \u003cem\u003eBeta\u003c/em\u003e sequences. After the alignment, specific \u003cem\u003eHs4\u003c/em\u003e or \u003cem\u003eBvHs4\u003c/em\u003e sequences were generated for each accession sequenced. The CLC-incorporated tool was used for protein translation, generating the corresponding polypeptide sequences.\u003c/p\u003e \u003cp\u003eFurthermore, we searched reference sequences from the susceptible beet lines EL10_2 (McGrath et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and W357B v1.0 (Dorn \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and the nematode-tolerant \u003cem\u003eB. vulgaris\u003c/em\u003e line U2Bv (Sielemann et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). Reference sequences from the \u003cem\u003eB. patula\u003c/em\u003e accession Bpat-1.0 (Rodr\u0026iacute;guez del R\u0026iacute;o et al. 2019), the \u003cem\u003eB. vulgaris\u003c/em\u003e ssp. \u003cem\u003emaritima\u003c/em\u003e accession Bmar1.0 (Rodr\u0026iacute;guez delR\u0026iacute;o et al. 2019) and a first \u003cem\u003eP. procumbens\u003c/em\u003e draft genome sequence (USDA_Ppro_WB292_v1.0, NCBI Genome Accession No. JBMGQN000000000, derived from USDA NPGS Accession Ames 4464). Using MEGA11 (Tamura et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), the sequences were aligned via the MUSCLE algorithm, and a maximum-likelihood phylogenetic tree (500 bootstraps) was generated. Alignments were visualized using pyBoxshade v. 1.2 (mdbaron42 \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe blastn suite (Camacho et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) of the National Center for Biotechnology Information (Sayers et al. 2022) was deployed for DNA sequence comparisons. The localization and conformation of single polypeptides were predicted using DeepLoc2.1 (\u0026Oslash;dum et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and DeepTMHMM (Hallgren et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), respectively.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vivo\u003c/em\u003e nematode infection assay\u003c/p\u003e \u003cp\u003eNematode infection tests were performed essentially as described by Lange and De Bock (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). The \u003cem\u003eH. schachtii\u003c/em\u003e strain 'Schach 0' (M\u0026uuml;ller \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) was propagated on susceptible sugar beet or rapeseed in sand-filled tubes in a greenhouse under LD conditions. Larvae were hatched from cysts in 3 mM ZnCl\u003csub\u003e2\u003c/sub\u003e in a Baermann-funnel apparatus. Before inoculation, the J2 larvae were concentrated to the desired density of 150 per ml. Plants were grown in plastic tubes filled with quartz sand and inoculated with 300 J2 larvae. Developing females were identified four weeks later.\u003c/p\u003e \u003cp\u003eExpression analysis\u003c/p\u003e \u003cp\u003eThe leaf and root samples of three biological replicates per accession were taken for the initial expression test. For the spatiotemporal expression analysis, samples of plant roots (R), hypocotyls (H), cotyledons (C), and leaves (L) of three biological replicates were taken shortly after germination (R, H, C) upon the development of the first true leaves (R, C, L), 21 days after germination (R, L), before inoculation (R, L), and 3 and 28 days post-inoculation (dpi; R, L). The samples were frozen in liquid nitrogen, and total RNA was isolated using the Universal RNA Kit (roboklon, Berlin, Germany), according to the manufacturer's instructions. The RNA quality was checked using a NanoDrop2000 spectrophotometer (ThermoFisher Scientific\u0026trade;) and agarose gel electrophoresis (2% gel, 100V, 12 min).\u003c/p\u003e \u003cp\u003e100 ng of RNA were incubated with 1 U DNase (ThermoFisher Scientific\u0026trade;) at 37\u0026deg;C for 30 min. Subsequently, the DNase was inactivated by adding 1 \u0026micro;l EDTA (50 mM), followed by incubation at 65\u0026deg;C for 10 min. Then, the cDNA was generated using the First Strand cDNA Synthesis Kit (ThermoFisher Scientific\u0026trade;), following the manufacturer's instructions. Using the \u003cem\u003eGAPDH\u003c/em\u003e primer combination for \u003cem\u003eBeta\u003c/em\u003e (BvGAPDH_F/BvGAPDH_R, Supplementary Table\u0026nbsp;2) or \u003cem\u003ePatellifolia\u003c/em\u003e (AS_BvPp_GAPDH_F/AS_BvPp_GAPDH_R, Supplementary Table\u0026nbsp;2) species, the cDNA quality was assessed by PCR.\u003c/p\u003e \u003cp\u003eFollowing the manufacturer's instructions, two \u0026micro;l cDNA were used for quantitative reverse transcription PCR (RT-qPCR) deploying the Platinum\u0026trade; SYBR\u0026trade; Green qPCR SuperMix-UDG (Invitrogen\u0026trade;, ThermoFisher Scientific\u0026trade;). The \u003cem\u003eGAPDH\u003c/em\u003e gene was used for gene expression normalization. For the detection of \u003cem\u003eHs4\u003c/em\u003e transcripts, the primer combinations AK_F7/AK_R5 and AS_BvHs4_F1/AS_BvHs4_R7 (Supplementary Table\u0026nbsp;2) were used. The obtained Ct-values were evaluated using the Pfaffl method (Pfaffl 2001).\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eNormally distributed data were statistically analyzed by a one-way analysis of variance (ANOVA) using Microsoft Excel. The non-normally distributed data were statistically analyzed by the Kruskal-Wallis rank sum test, followed by a Hochberg-corrected Dunn post-hoc test using the online calculator Astatsa (Vasavada \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and the open-source statistical computing software R version 4.3.3 (R Core Team \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) using the package \"PMCMRplus\" (Pohlert \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eData availability statement\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eP. procumbens\u003c/em\u003e sequence data generated and analysed during the current study are available in GenBank of the NCBI (https://www.ncbi.nlm.nih.gov/) repository (Genome Accession No. JBMGQN000000000). The \u003cem\u003eHs4\u003c/em\u003e homologs sequences are available in GenBank of the NCBI (GenBank Accession Numbers PV258691 to PV258713) (Supplementary Table 6).\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eWe thank Birgit Defant, Maike Schneider, S. Greve, and I. Baumgartner for their technical assistance. We thank the Institute of Clinical Molecular Biology in Kiel for providing Sanger sequencing, as partly supported by the DFG Cluster of Excellence \u0026quot;Inflammation at Interfaces\u0026quot; and \u0026quot;Future Ocean.\u0026quot;\u003c/p\u003e\n\u003cp\u003eAuthor contribution\u003c/p\u003e\n\u003cp\u003eAS designed and performed the experiments, followed by analyzing the data. CJ led the design of the study and supervised data analysis. KD contributed unpublished sequence data. AS drafted the manuscript, which CJ and KD revised. All authors read and approved the final article. Correspondence and requests for materials should be addressed to CJ.\u003c/p\u003e\n\u003cp\u003eConflict of interest\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAspden JL, Wallace EW, Whiffin N (2023) Not all exons are protein coding: Addressing a common misconception. Cell Genomics 3:100296. https://doi.org/10.1016/j.xgen.2023.100296\u003c/li\u003e\n\u003cli\u003eCamacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) BLAST+: architecture and applications. BMC Bioinformatics 10:421. https://doi.org/10.1186/1471-2105-10-421\u003c/li\u003e\n\u003cli\u003eCurtis GJ (1968) Observations of fruit shape and other characters in the species of the section \u003cem\u003ePatellares\u003c/em\u003e, genus \u003cem\u003eBeta\u003c/em\u003e. Euphytica 17:485\u0026ndash;491. https://doi.org/10.1007/BF00056252\u003c/li\u003e\n\u003cli\u003eDaub M (2021) The beet cyst nematode (\u003cem\u003eHeterodera schachtii \u003c/em\u003e): an ancient threat to sugar beet crops in Central Europe has become an invisible actor. In: Sikora RA, Desaeger J, Molendijk L (eds) Integrated nematode management: state-of-the-art and visions for the future, 1st edn. CABI, UK, pp 394\u0026ndash;399. https://doi.org/10.1079/9781789247541.0055\u003c/li\u003e\n\u003cli\u003eDohm JC, Minoche AE, Holtgr\u0026auml;we D et al (2014) The genome of the recently domesticated crop plant sugar beet (\u003cem\u003eBeta vulgaris\u003c/em\u003e). Nature 505:546\u0026ndash;549. https://doi.org/10.1038/nature12817\u003c/li\u003e\n\u003cli\u003eDorn K (2022) \u003cem\u003eBeta vulgaris \u003c/em\u003eW357B genome. Zenodo. https://zenodo.org/records/5911852. Accessed 21 June 2024. https://doi.org/10.5281/zenodo.5911852\u003c/li\u003e\n\u003cli\u003eFreeman M (2009) Rhomboids: 7 years of a new protease family. Seminars in Cell \u0026amp; Developmental Biology 20:231\u0026ndash;239. https://doi.org/10.1016/j.semcdb.2008.10.006\u003c/li\u003e\n\u003cli\u003eFrese L, B\u0026uuml;low, Castro S, Duarte C, Iriondo JM, Lohwasser U, Loureiro J, Maxted N, Nachtigall M, Nobrega H, Pinheiro de Carvalho, M. \u0026Acirc;. A., Santos Guerra A, Romeiras MM, Rubio ML, Rey E (2017) Genetic diversity of \u003cem\u003ePatellifolia \u003c/em\u003especies (GeDiPa)\u003c/li\u003e\n\u003cli\u003eFrese L, Nachtigall M, Iriondo JM, Rubio Teso ML, Duarte MC, Pinheiro de Carvalho, Miguel \u0026Acirc;ngelo A. (2019) Genetic diversity and differentiation in \u003cem\u003ePatellifolia \u003c/em\u003e(Amaranthaceae) in the Macaronesian archipelagos and the Iberian Peninsula and implications for genetic conservation programmes. 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PloS One 11:e0152456. https://doi.org/10.1371/journal.pone.0152456\u003c/li\u003e\n\u003cli\u003eSavitsky H (1975) Hybridization between \u003cem\u003eBeta vulgaris \u003c/em\u003eand \u003cem\u003eB. procumbens \u003c/em\u003eand transmission of nematode (\u003cem\u003eHeterodera schachtii\u003c/em\u003e) resistance to sugar beet. Canadian Journal of Genetics and Cytology 17:197\u0026ndash;209. https://doi.org/10.1139/g75-027\u003c/li\u003e\n\u003cli\u003eScott AJ, Ford-Lloyd BV, Williams JT (1977) \u003cem\u003ePatellifolia\u003c/em\u003e, nomen novum (Chenopodiaceae). Taxon 26:284. https://doi.org/10.2307/1220567\u003c/li\u003e\n\u003cli\u003eSielemann K, Pucker B, Orsini E, Elashry A, Schulte L, Vieh\u0026ouml;ver P, M\u0026uuml;ller AE, Schechert A, Weisshaar B, Holtgr\u0026auml;we D (2023a) Genomic characterization of a nematode tolerance locus in sugar beet. BMC Genomics 24:748. https://doi.org/10.1186/s12864-023-09823-2\u003c/li\u003e\n\u003cli\u003eSielemann K, Schmidt N, Guzik J, Kalina N, Pucker B, Vieh\u0026ouml;ver P, Breitenbach S, Weisshaar B, Heitkam T, Holtgr\u0026auml;we D (2023b) Pangenome of cultivated beet and crop wild relatives reveals parental relationships of a tetraploid wild beet. bioRxiv. https://www.biorxiv.org/content/10.1101/2023.06.28.546919v1. Accessed 5 March 2024. https://doi.org/10.1101/2023.06.28.546919\u003c/li\u003e\n\u003cli\u003eSielemann K, Pucker B, Schmidt N, Vieh\u0026ouml;ver P, Weisshaar B, Heitkam T, Holtgr\u0026auml;we D (2022) Complete pan-plastome sequences enable high resolution phylogenetic classification of sugar beet and closely related crop wild relatives. BMC Genomics 23:113. https://doi.org/10.1186/s12864-022-08336-8\u003c/li\u003e\n\u003cli\u003eSijmons PC, Atkinson HJ, Wyss U (1994) PARASITIC STRATEGIES OF ROOT NEMATODES AND ASSOCIATED HOST CELL RESPONSES. Annual Review of Phytopathology 32:235\u0026ndash;259. https://doi.org/10.1146/annurev.py.32.090194.001315\u003c/li\u003e\n\u003cli\u003eTamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Molecular Biology and Evolution 38:3022\u0026ndash;3027. https://doi.org/10.1093/molbev/msab120\u003c/li\u003e\n\u003cli\u003eUrban S, Dickey SW (2011) The rhomboid protease family: a decade of progress on function and mechanism. Genome Biology 12:231. https://doi.org/10.1186/gb-2011-12-10-231\u003c/li\u003e\n\u003cli\u003eVaradi M, Bertoni D, Magana P et al (2024) AlphaFold Protein Structure Database in 2024: providing structure coverage for over 214 million protein sequences. Nucleic Acids Research 52:D368-D375. https://doi.org/10.1093/nar/gkad1011\u003c/li\u003e\n\u003cli\u003eVaradi M, Anyango S, Deshpande M et al (2022) AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Research 50:D439-D444. https://doi.org/10.1093/nar/gkab1061\u003c/li\u003e\n\u003cli\u003eVasavada N (2016) Astatsa - Online Web Statistical Calculators. https://astatsa.com/\u003c/li\u003e\n\u003cli\u003eViglierchio DR (1960) Resistance in \u003cem\u003eBeta \u003c/em\u003especies to the sugar beet nematode, \u003cem\u003eHeterodera schachtii\u003c/em\u003e. Experimental Parasitology 10:389\u0026ndash;395. https://doi.org/10.1016/0014-4894(60)90075-8\u003c/li\u003e\n\u003cli\u003eWagner H, Gimbel E-M, Wricke G (1989) Are \u003cem\u003eBeta procumbens \u003c/em\u003eChr. Sm. and\u003cem\u003e Beta webbiana \u003c/em\u003eMoq. Different Species? Plant Breeding 102:17\u0026ndash;21. https://doi.org/10.1111/j.1439-0523.1989.tb00309.x\u003c/li\u003e\n\u003cli\u003eWilliams JT, Scott AJ, Ford-Lloyd BV (1976) Patellaria: A new genus in the Chenopodiaceae. Feddes Repertorium 87:289\u0026ndash;292. https://doi.org/10.1002/fedr.19760870502\u003c/li\u003e\n\u003cli\u003eWyss U (2002) Feeding Behavior of Plant-Parasitic Nematodes. In: Lee D (ed) The Biology of Nematodes. CRC Press, pp 233\u0026ndash;260. https://doi.org/10.1201/b12614-10\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Materials","content":"\u003cp\u003eSupplementary Materials are not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Beta vulgaris, Patellifolia, Heterodera schachtii, ER, sugar beet, plant parasitic nematodes","lastPublishedDoi":"10.21203/rs.3.rs-6653794/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6653794/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlant-parasitic nematodes are economically important threats to global crop production. The beet cyst nematode (\u003cem\u003eHeterodera schachtii\u003c/em\u003e) is a crucial pest in sugar beet (\u003cem\u003eBeta vulgaris\u003c/em\u003e). While all species of the genus \u003cem\u003eBeta\u003c/em\u003e are highly susceptible, the three species of the beet wild relative genus \u003cem\u003ePatellifolia\u003c/em\u003e are entirely resistant. Recently, we cloned the \u003cem\u003eHs4\u003c/em\u003e gene from \u003cem\u003eP. procumbens\u003c/em\u003e, which confers complete resistance. In this study, we aimed to determine whether putative \u003cem\u003eHs4\u003c/em\u003e orthologs exist in \u003cem\u003eBeta\u003c/em\u003e and \u003cem\u003ePatellifolia\u003c/em\u003e species. The \u003cem\u003eHs4\u003c/em\u003e gene consisted of 4999 bp, with six exons and five introns. \u003cem\u003ePatellifolia\u003c/em\u003e species contain highly similar \u003cem\u003eHs4\u003c/em\u003e homologs. Single nucleotide polymorphisms and insertions/deletions between the accessions and species could be detected. We found an exonic integration of three bases, resulting in the addition of one amino acid. Interestingly, this variant was present in single accessions of all three \u003cem\u003ePatellifolia\u003c/em\u003e species. \u003cem\u003eBeta vulgaris\u003c/em\u003e contains an \u003cem\u003eHs4\u003c/em\u003e homolog (\u003cem\u003eBvHs4\u003c/em\u003e) with 60% protein identity to Hs4. \u003cem\u003eBvHs4\u003c/em\u003e homologs were present in all \u003cem\u003eBeta\u003c/em\u003e species analyzed. Further, we examined the expression patterns of \u003cem\u003eHs4\u003c/em\u003e and \u003cem\u003eBvHs4\u003c/em\u003e homologs. While \u003cem\u003eHs4\u003c/em\u003e homologs from \u003cem\u003ePatellifolia\u003c/em\u003e species are strongly expressed in roots, \u003cem\u003eBvHs4\u003c/em\u003e homologs are expressed mainly in leaves. When the spatio-temporal expression of \u003cem\u003eHs4\u003c/em\u003e was examined, no response to nematode inoculation was observed. These results are highly relevant for searching for functional \u003cem\u003eHs4\u003c/em\u003e alleles and breeding nematode-resistant varieties.\u003c/p\u003e","manuscriptTitle":"Subcellular localization and differential expression account for the function of the nematode resistance gene Hs4","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-18 03:00:55","doi":"10.21203/rs.3.rs-6653794/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-09T13:30:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-04T15:04:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-23T09:38:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"147649803827952152129080524755686223656","date":"2025-06-16T11:55:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"65137769768520595770084621676588544425","date":"2025-06-16T00:38:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-13T12:47:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-08T12:33:57+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-27T14:32:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-26T08:30:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-13T09:11:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d81701f5-954d-47af-9a01-48890342c7f6","owner":[],"postedDate":"June 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50118007,"name":"Biological sciences/Genetics/Agricultural genetics"},{"id":50118008,"name":"Biological sciences/Genetics/Plant breeding"}],"tags":[],"updatedAt":"2026-03-02T16:02:25+00:00","versionOfRecord":{"articleIdentity":"rs-6653794","link":"https://doi.org/10.1038/s41598-026-40666-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-02-28 15:59:01","publishedOnDateReadable":"February 28th, 2026"},"versionCreatedAt":"2025-06-18 03:00:55","video":"","vorDoi":"10.1038/s41598-026-40666-5","vorDoiUrl":"https://doi.org/10.1038/s41598-026-40666-5","workflowStages":[]},"version":"v1","identity":"rs-6653794","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6653794","identity":"rs-6653794","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}