Divergent molecular pathways govern temperature-dependent wheat stem rust resistance genes Sr6, Sr13 and Sr21

Divergent molecular pathways govern temperature-dependent wheat stem rust resistance genes Sr6, Sr13 and Sr21 | 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 Divergent molecular pathways govern temperature-dependent wheat stem rust resistance genes Sr6, Sr13 and Sr21 Peng Zhang, Tim Hewitt, Keshav Sharma, Jianping Zhang, Prabin Bajgain, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4674841/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 May, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract The wheat stem rust pathogen, Puccinia graminis f. sp. tritici ( Pgt ), has caused devastating crop losses worldwide. Several stem rust resistance ( Sr ) genes display temperature-dependent immune responses. Sr6 -mediated resistance is enhanced at lower temperatures whereas Sr13 and Sr21 resistances are enhanced at higher temperatures. Here we report cloning of Sr6 by mutagenesis and resistance gene enrichment and sequencing (MutRenSeq), showing it to encode an NLR protein with an integrated BED domain. Sr6 temperature sensitivity was also transferred to wheat plants transformed with the Sr6 transgene. Differential gene expression analysis using near-isogenic wheat lines inoculated with Pgt at varying temperatures revealed that genes upregulated in the low-temperature-effective Sr6 response differed significantly from those upregulated in the high-temperature-effective responses associated with Sr13 and Sr21 . Understanding the molecular mechanisms and pathways involved in temperature sensitivity can inform future strategies for deployment and engineering of genetic resistance in response to a changing climate. Biological sciences/Genetics/Plant genetics Biological sciences/Molecular biology NLR Puccinia graminis temperature sensitivity Triticum spp Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Wheat stem rust, caused by the fungal pathogen Puccinia graminis f. sp. tritici ( Pgt ), poses a significant threat to global wheat production. Virulent strains have overcome widely deployed disease resistance genes, including Sr6 1 . This gene, once widely effective, is now widespread and found in about 13% of global spring wheat varieties 2 . Stem rust epidemics have historically caused severe damage during warm weather, affecting continental-scale wheat production 3,4 . In the United States, stem rust outbreaks from 1919 to 1954 led to substantial statewide losses 5 often causing up to 50% yield loss 3 . The release of the Sr6 -bearing cultivar Selkirk was particularly successful in combatting stem rust epidemics that swept across North America from the 1920s to 1960s. The first widely grown Australian stem rust resistant cultivar was Eureka carrying Sr6 and released in 1938. However, as early as in 1942, virulent Pgt races were identified, and the cultivar went through a series of recurrent epidemics before it was removed from cultivation after the mid-1960s 6 . Nevertheless, Sr6 still provides resistance in some parts of North America 7,8 and possibly India 9 , particularly when combined with other genes such as Sr57 ( Lr34 ) 10 or Sr2 (as was the case with Selkirk). The emergence of Pgt races TTKSK (commonly referred to as Ug99) and TKTTF (‘Digalu’ race) in East Africa was particularly alarming. These races were virulent to widely deployed stem rust resistance genes, rendering 80–90% of global wheat varieties susceptible 11–15 . Spread across multiple regions, the Ug99 and Digalu races and variants overcame resistance genes such as Sr24 , Sr36 , and SrTmp 11–13,16 . Plants employ basal and resistance (R)-mediated defense responses to infection by microbial pathogens 17 . Both responses are influenced by heat stress 18 , highlighting the adaptability of the plant defense system to varying temperatures. Pathogen-Associated Molecular Patterns (PAMPs) activate PAMP-triggered immunity (PTI) upon detection. Host-adapted pathogens manipulate PTI using effector proteins, which can be recognized by plant intracellular receptors, leading to Effector-Triggered Immunity (ETI). ETI, induced by host R genes, results in hypersensitive responses or programmed cell death 18 . Durable disease resistance is essential to safeguard wheat from ever-changing rust pathogens 19 . Stem rust resistance is often placed in two categories: Adult plant resistance (APR), conferred by multi-pathogen resistance genes such as Sr2, Sr57 / Lr34 Sr58 / L46 and Sr55 / Lr67 (and other designations) that show effectiveness at later growth stages, and all-stage resistance (ASR), effective from the seedling stage. Cloned ASR genes for stem rust include Sr22, Sr26, Sr33, Sr35, Sr45 , Sr50 , Sr61 , and Sr62 as well as the temperature sensitive Sr13 and Sr21 20–26 . These R genes typically encode coiled coil-nucleotide binding leucine-rich repeat (CC-NBS-LRR or NLR) proteins or protein kinases, such as Sr60 27 . Notably, Sr13 and Sr21 are more effective at higher temperatures 1,11,23,24,28 , in contrast to Sr6 which is more effective below 20°C and ineffective above 24–27°C 29,30 . Similarly, stem rust resistance genes Sr10, Sr15 , and Sr17 are less effective at higher temperatures 24 . Plants susceptible at a non-permissive temperature regained resistance once the temperature returned to a permissive level 31 . Light conditions also impact the resistance response 32 . Whereas Sr13 and Sr21 were previously cloned 23,24 , here we report the cloning of Sr6 using mutagenesis and R gene enrichment and sequencing (MutRenSeq) and show that it is an NLR integrated with a non-canonical zinc finger BED domain. Stable transformation confirmed Sr6 identity which induced a characteristic temperature-sensitive resistance phenotype in transgenic plants challenged with an Sr6- avirulent Pgt race. RNA sequence analysis showed that gene expression was affected by temperature. Differential gene expression analysis on near-isogenic wheat lines carrying either Sr6 , Sr13 or Sr21 elucidated varied defence pathways in response to different temperatures. Results Sr6 candidate isolated by MutRenSeq and verified in recombinant inbred lines To clone Sr6 , we identified susceptible EMS-generated mutants from the substitution line Chinese Spring*5/Red Egyptian 2D (CS/RE 2D) produced by Sears et al. 33 . Seven independent mutants together with wild-type CS/RE 2D were processed using the MutRenSeq pipeline. Captured sequencing reads from these seven mutants and the wild type (WT) were aligned to a de novo reference assembly of the WT reads. One contig (#733836) of 2,842 bp containing a SNP was identified in three of the seven mutants and possessed NB-ARC and LRR motifs but no CC motif. A second contig (#737511) of 2,218 bp containing a SNP in a different three mutants was identified and possessed only LRR motifs. The last ~ 600 bp of contig #733836 had homology to the first ~ 600 bp of contig #737511 suggesting they overlapped but were not joined during assembly possibly due to low coverage and/or ambiguity of reads covering the bridging region. To confirm that these sequences were physically joined, a second assembly was generated based on a combined pool of reads from all the mutants and WT. The second assembly produced a scaffold (#2265) that represented a joining of the two contigs in six of the seven mutants having SNPs (Supplementary Fig. 1A). No SNP was detected in mutant 3981-4, but since scaffold #2265 did not contain any CC domain-encoding motifs, mutant 3981-4 was predicted to harbour a mutation in a potentially missing upstream sequence. Scaffold #2265 was aligned to the Chinese Spring RefSeq v1.0 (CSv1) reference assembly 34 using BLAST, with the top hit (~ 91%) to chromosome 2D approximately 5.1 Mbp from the Sr6 -proximal marker Xwmc453 identified by Tsilo et al. 32 . The matching sequence overlapped with high-confidence annotated gene TraesCS2D02G111500 , encoding a disease resistance protein. A dominant PCR marker Sr6STS1 designed based on the sequence of scaffold #2265 showed specificity to Sr6 -carrying lines CS/RE 2D and Manitou (Supplementary Fig. 2A). Additionally, a PCR product spanning almost the full length of scaffold #2265 confirmed linkage of the two initially discovered contigs (Supplementary Fig. 2B) and was amplified from the six mutants with SNPs. Sanger sequencing confirmed presence of the SNP mutations. Each of the six mutants contained a nonsynonymous SNP, with five resulting in missense mutations and one in a nonsense mutation (Fig. 1 A). 5’RACE (rapid amplification of cDNA ends) from WT RNA revealed the presence of an additional 5’ sequence that encoded both CC and zinc finger BED domains. Primers were designed to amplify this additional 5’ region from DNA of mutant 3981-4 (Supplementary Fig. 2C), from which a mutation had yet to be identified. Sanger sequencing revealed the presence of a G-to-A SNP (Supplementary Fig. 1B) in the 5’UTR, 20 bp upstream of the first start codon. Although this mutation does not alter the coding sequence, mutations in the 5’UTR are known to impact translation 35,36 . Overall, point mutations were confirmed in the candidate sequence for all seven mutants (Supplementary Table 1). To further verify the candidate as Sr6 , a population of 197 F 3 lines from a reciprocal cross of CS and CS/RE 2D segregating for Sr6 were screened for response to Pgt race 21 − 2,3,7. The dominant STS marker Sr6STS1 , based on the candidate sequence, was present in all homozygous resistant and segregating lines, and absent in homozygous susceptible lines. Full gene structure of Sr6 obtained from whole genome sequenced wheat accessions The Sr6 candidate sequence compared against published wheat genome assemblies 37 had a 100% match on chromosome 2D of the genome assembly of cv. Landmark (Supplementary Fig. 3). The full upstream and downstream sequences from Landmark presented an opportunity to use the native regulatory elements for transgenic experiments with Sr6 . However, the probable promoter region, less than 2 kb upstream of the putative start of codon, contained a large gap in sequence denoted by Ns (unknown bases). Fortunately, the Sr6 sequence was also found in an assembled scaffold of cv. Claire harbouring a gapless upstream sequence. The sequence from Claire was used to close the gap present in the Landmark sequence (Supplementary Fig. 4A). Additionally, a scaffold from cv. Robigus, which contained a non-identical coding sequence, did have an identical upstream region to Claire, indicating the sequence overlapping the gap was conserved. A transcript was identified from 5’ and 3’ RACE amplification having an intron/exon structure showing conservation with the CSv1 homologue TraesCS2D02G111500. Additionally, a complete transcript retrieved from the de novo transcriptome assembly of Avocet R supported conservation of the intron/exon structure at the 5’ end (Supplementary Fig. 3). Sr6 is diverged from known wheat NLRs but contains a conserved BED domain The Sr6 candidate was aligned to 16 reference genomes, of which, only Landmark and Claire contained a matching sequence. However, the ~ 165 bp BED domain encoding region (exon 2) appears conserved, as seen in the alignment with the Avocet R ortholog (Supplementary Fig. 3). The BED domain sequence was also identical in the chromosome 2D homologs in ArinaLrFor, Jagger, Julius, Lancer, Norin 61 and Robigus. The BED domains of the 2D homologs in CSv1 and Weebill 1, as well as the chromosome 2B homoeolog in Zavitan (tetraploid) had eight SNPs with Sr6 . The 2D homologs in Mace, SY Mattis and spelt accession PI190962 had 16 SNPs. The full length protein of SR6, its zinc ion binding region and ATP binding region were predicted by AlpahFold3 38 (Fig. 1 B). The BED domain of Sr6 was also compared to those of stripe rust R genes Yr5 and Yr7 , the only cloned wheat R genes thus far known to contain a BED domain 39 . Yr5 and Yr7 have the same BED-NB-LRR configuration as Sr6 and have a similar intron/exon arrangement in that each of the CC, BED and NB-LRR domains reside in its own exon. Whereas the protein sequence of the Sr6 candidate is quite differentiated from that of Yr5/Yr7 , 12 residues in the BED domain appear conserved (Supplementary Fig. 4B). The translated sequence of the Sr6 candidate was compared against the NCBI protein database but produced no significant hits (< 80% sequence identity). Comparison of the protein sequence of the candidate against a panel of known CNL class NLRs did not indicate any close relationship although it did cluster with other wheat R genes including Sr21 , Yr5 and Yr7 (Supplementary Fig. 5). A commonality between these three genes and Sr6 is that they reside on group 2 chromosomes, but they are not located at orthologous positions as Sr6 is in the 2DS arm whereas Sr21 and Yr5/Yr7 are in the 2AL and 2BL arms, respectively 24,39 . Transgenic complementation with the Sr6 candidate confers temperature-sensitive resistance segregating in T1 generation In addition to lines CS/RE 2D and Manitou, which are known to have Sr6 1 , several transformable varieties were tested with marker Sr6STS1 to screen for an appropriate transgene host. Wheat varieties Fielder and Yitpi were positive whereas Westonia and durum cultivar Stewart were negative (data not shown). Furthermore, phenotyping the seed stocks of Fielder maintained at CSIRO Canberra identified low infection types (ITs) with Sr6 -avirulent Pgt races 21 − 0 and 21 − 2,3,7, and high IT with Sr6 -virulent race 21 − 1,2,3,5,6,( 7 ) (University of Sydney Culture no. 50); Westonia had high ITs with all three races even though it was previously reported to carry Sr6 40 . Consequently, the CSIRO stock of Westonia was chosen as the susceptible host for transgenic complementation. Agrobacterium transformation of embryos of Westonia with the Sr6 candidate gene, including native regulatory elements, yielded two successful T 0 transformants. The T 1 progeny were grown at either low (18℃) or high (25℃) temperatures and were infected with Pgt race 21 − 0 isolate at two weeks old. The plants were scored for their resistance responses at 14 days post-inoculation (dpi) (Fig. 2 , Supplementary Fig. 6). At low temperature, there was clear distinction between resistant (n = 39) and susceptible (n = 8) individuals across both parental batches (~ 3:1 segregation overall, χ 2 = 1.60, p = 0.207) where resistance was observed as small necrotic lesions or small uredinia surrounded by necrosis. At high temperature, the infection types were much higher overall and there were 13 susceptible and 35 resistant individuals (~ 3:1 segregation overall, χ 2 = 0.11, p = 0.739). The presence of resistant T 1 plants confirmed resistance activity of the transgene and its identity as Sr6 . The higher ratio of susceptible plants at high temperature comports with expected loss of resistance seen with endogenous Sr6 at high temperature (Fig. 2 ) and suggests temperature sensitivity is mediated by the Sr6 gene itself. Apparently 25℃ was not sufficient to achieve a complete knockout of Sr6 with the genetic material and environmental conditions of the present experiment. All T 1 individuals were assayed with the Sr6 STS marker to check for co-segregation with resistance. Unexpectedly, all individuals were positive after repeated tests, suggesting one or more non-functional copies of the transgene integrated into the genome of all individuals. Position effect variegation of transgene expression among individuals may have led to segregation of resistant and susceptible phenotypes. Sr6 expression was measured by qPCR and the relative expression (RE) was calculated for each individual and distributions of RE values from resistant and susceptible groups were compared between high and low temperature conditions (Fig. 2 ). The difference between low-temperature resistant and high-temperature resistant groups was not significant (p = 0.2883 Welch’s t -test), indicating temperature generally had minimal impact on expression. Whereas resistant plants had a wide range of RE values at low temperature, all susceptible plants showed little to no expression. In contrast, a considerable number of ‘susceptible’ [moderately resistant/moderately susceptible (MRMS) and MS] plants (n = 10) maintained relatively high levels of expression at high temperature, suggesting that suppression of resistance in these plants was not solely modulated by expression. The difference between low-temperature susceptible and high-temperature susceptible was significant (p = 0.0019) (Fig. 4 ). Unsurprisingly, the expression difference between resistant and susceptible groups was very high (p < 0.0001) at both temperatures. Sr gene expression tracks with resistance response at different temperatures The NILs LMPG- Sr6 , LMPG- Sr13 and LMPG-Sr 21 were inoculated with Pgt race MCCFC (isolate no 59KS19) and sampled at different timepoints. The Sr6 NIL was highly resistant (IT ‘0’) at low temperature (18℃) but susceptible (IT ‘3 + C’) at high temperature (25℃) (Fig. 3 B). Sr13 and Sr21 showed the opposite pattern where the corresponding lines were more resistant at the higher temperature (IT ‘2’ and ‘2-’, respectively) compared to the low temperature (IT ‘2 + 3’ and ‘2+’, respectively) (Figs. 3 C, 3 D). Sr6 was upregulated at low temperature (Supplementary Fig. 7.1). Specifically, the level of Sr6 expression was higher at day 3 at low temperature but was lower at high temperature. At low temperature, Sr13 was upregulated at day 1 but downregulated at day 3, whereas it was downregulated at day 1 and upregulated at day 3 at high temperature. Sr21 was upregulated on both day 1 and day 3 under both low and high temperature conditions. Biotic stress-related genes show differential expression between Sr lines at high and low temperatures Under conditions leading to more resistant responses (high temperature for Sr13 and Sr21 ; low temperature for Sr6 ), six pathogenesis related (PR) and four jasmonic acid (JA) pathway genes were tested by qPCR for gene expression (Supplementary Figs. 7.2–7.4, Supplementary Tables 5.1, 5.2). All were upregulated in Sr6 lines under low temperature, except PR4 which was downregulated on day 3. PR1 and PR9 were highly expressed at day 3 in Sr6 lines. Most of the PR genes were upregulated in Sr13 lines under high temperature except PR3 , PR4 and PR5 which were downregulated at day 3 (Supplementary Fig. 7.3A). In Sr21 lines under high temperature, PR3 and PR4 were downregulated at day 1 whereas PR1 , PR2 , PR5 and PR9 were upregulated (Supplementary Fig. 7.4A). All PR genes were downregulated in Sr21 lines at day 3. Expression of PR genes relates to the salicylic acid (SA) pathway and is known to be associated with resistance against pathogen attack 23 . Similarly, relative expression of several components of the jasmonic acid signalling were tested under high and low temperatures. Among the four tested JA related genes in Sr6 lines, three were downregulated under low temperature except JA3 , which was upregulated at day 1 (Supplementary Fig. 7.2B). In Sr13 lines, JA1 and JA3 were upregulated under high temperature at day 1, but all JA genes were downregulated in Sr13 lines on all other conditions (Supplementary Fig. 7.3B). In Sr21 lines, JA related genes were downregulated (Supplementary Fig. 7.4B) RNAseq analysis shows divergence in broader expression patterns between Sr lines Differential expression of most genes (35,000–36,000) was non-significant using our analysis criteria. RNAseq analysis indicated that 57 to 872 genes were upregulated in the NILs compared to parental LMPG at both low and high temperatures (Supplementary Fig. 8). Eight genes uniquely expressed in all three lines, i.e. four upregulated genes; Aquaporin PIP1mRNA (PIP1), Oxygen-evolving enhancer protein-2 (OEE-2), Chlorophyll binding protein (CBP) and Alpha-amylase/trypsin inhibitor-like protein (AAMY) at day 3 were validated through qPCR analysis (Supplementary Table 5.3). The qPCR results validated the results obtained from RNAseq analysis (Supplementary Fig. 7.5). OEE-2 and CBP were upregulated in LMPG- Sr6 under low temperature but were downregulated under high temperature. PIP1 was downregulated under both low and high temperatures. OEE-2 was downregulated in LMPG- Sr13 under high and low temperature whereas it was upregulated under high temperature in LMPG- Sr21. PIP1 was upregulated in LMPG- Sr13 and LMPG- Sr21 and downregulated in LMPG- Sr6 . Additionally, AAMY was upregulated under both high and low temperatures in LMPG- Sr21 . CBP was downregulated under high temperature but upregulated under low temperature in LMPG- Sr21 as in LMPG- Sr6 . This indicated that CBP showed a similar expression pattern for both a low- and a high-temperature-sensitive Sr gene. Most DEGs in LMPG- Sr13 and LMPG- Sr21 followed a similar expression pattern that differed from LMPG- Sr6 . The number of DEGs for a particular NIL under a certain temperature ranged from 26 to 553 (Supplementary Fig. 9). Most of the transcripts upregulated in LMPG- Sr6 under low temperature (CR) were distributed across the genome, but with a higher number of genes mapped to chromosomes 2B and 3B on both day 1 and day 3 (Supplementary Fig. 10). Similarly, DEGs mapped to most chromosomes in LMPG- Sr13 and LMPG- Sr21 , with some variation where reads tended to map frequently to chromosomes 1A, 2B, and homoeologous group 7 members. Gene ontology (GO) analyses were conducted using GeneOntology and ShinyGo (Supplementary Figs. 10–13, 17–20; Supplementary Tables 2–4). Enrichment analysis in Sr6 lines demonstrated that genes related to defence response to fungal infection, response to biotic stimulus, and various pathogen resistance proteins were expressed under low temperature. These pathways corresponded with the strong upregulation of PR genes for Sr6 at low temperature. On the contrary, significant pathways were not observed for LMPG- Sr13 under low temperature on day 1. However, when at the high temperature where Sr13 is most effective, numerous pathways including ribosomal small subunit assembly, carton utilization, and photosynthesis were upregulated. Upregulated genes included OEE-2 and Cytochrome C. For LMPG- Sr21 proton transmembrane transport was upregulated on day 1 at both high and low temperatures. Numerous differences in upregulated pathways were observed at day 3 where various biosynthetic and metabolic processes were upregulated at high temperature, but not low temperature. Genes upregulated on day 3 included kinase proteins, an ABC transporter, OEE-2, and several membrane trafficking proteins. Protein structural comparison and analysis To further investigate any potential association between temperature-sensitive resistance exhibited by SR6, SR13, and SR21, we performed in-silico protein structure modelling using AlphaFold3 38 . Full-length protein structure prediction of all three proteins clearly displayed three subdomains: an N terminal domain, an NB-ARC domain, and an LRR domain with overall high confidence (Supplementary Figs. 14A, 15). Although all three full length structures are well conserved in protein subdomain composition, the N terminal domains of SR6 and SR21 are significantly different from SR13 that contains a four helix bundle to comprise a coiled coil structure (Supplementary Fig. 14B), whereas the N terminal subdomain of SR6 formed by four helices with a BED domain motif insert between a3 and a4, the N terminal subdomain of SR21 appears to form an extra helix in addition to the four helices coiled coil bundle (Supplementary Fig. 14C). Nevertheless, despite all these distinctive N terminal structures, all three proteins seem to have a highly conserved first three helices bundle and noticeably the a3 helix is always the helix that is physically most adjacent to the LRR domain (Supplementary Fig. 14C). We further compared the zinc finger BED motif from SR6 with other zinc finger BED motifs from other BED domain-containing resistance proteins. All zinc finger BED structures from SR6, YR5a, RPH15, and XA1 highly resembled each other with pairwise Root-Mean-Square Deviation (RMSD) value less than 1 (Supplementary Fig. 16A), despite the rather low sequence similarity (Supplementary Fig. 16B). The WebLogo diagram unsurprisingly displayed a high conservation only for cysteines and histidines, which are predicted to bind to the zinc ions (Zn 2+ ) and resemble a “finger” shape (Supplementary Fig. 16C). Overall, we did not identify any obvious correlation between the resistance temperature sensitivity and the protein structure composition of SR6, SR13, and SR21. Discussion Mourad et al. 8 identified 32 Sr6 -associated SNPs that intersected functional gene annotations in Chinese Spring. However, none of them overlapped with the candidate we identified in this study. We isolated Sr6 using MutRenSeq. After Yr5/YrSP and Yr7 , Sr6 is the fourth all-stage cloned wheat rust R gene containing a BED domain. However, such domain integrations are not uncommon in angiosperms. The rice resistance gene Xa1 was identified as a BED-containing NLR 41 and recent genomic analyses identified many NLRs that incorporate non-canonical, integrated domains (IDs) 42–44 . The BED domain does appear to be required for resistance as Marchal et al. 39 noted that a single, induced SNP in the BED domain of Yr7 led to loss of resistance. Nevertheless, little is known about the functional role of BED domains in immune receptors despite a thorough genome-wide comparative evolutionary analysis of zinc finger BED transcription factor genes in land plants 45 . In Arabidopsis, the non-NB-LRR gene DAYSLEEPER encodes a BED domain that was shown to bind DNA 46 . In the context of immunity, IDs are known to act as decoys for pathogen effectors. For example, an integrated WRKY domain in Arabidospsis R gene RRS1-R binds bacterial effectors that target WRKY transcription factors 47 . It is unclear whether BED domains serve a similar purpose in ETI. However, the identification of a BED domain within leaf rust resistance gene Rph15 from barley suggests that different rust pathogen species adapted to distinct hosts may have effectors targeting similar transcription factor domains 48 . The BED domain in Sr6 appeared to be conserved across numerous accessions, not unlike the CC domain, so a potential role in signalling cannot be ruled out. Our in-silico structural modelling and analysis did not show an association between the full-length structures or diverse N terminal structures of SR6, SR13, SR21 and temperature sensitivity of resistance. Whereas Sr13 encodes a classic CC-NB-LRR, Sr21 and the SR9 allelic series encode NLR proteins with a unique N terminus comprised of five helices bundle 49 , and Sr6 falls into the zinc finger BED domain containing R protein encoding gene subset. Interestingly, we observed that all α3 helices from these proteins were the closest unit to their respective LRR domains. It is consistent with previous studies showing that the EDVID motif in the α3 helix from the CC domain of CNL protein SR35 interacts with its LRR domain to form a conserved EDVID-LRR R−cluster interface. It is considered as an evolutionarily conserved stabilization mechanism of CNL resistosomes and is important in mediating resistance-induced cell death 50 . Our observation implies both the zinc finger BED-containing group (SR6, YR5a, RPH15, XA1) and SR21 and the SR9 group could potentially share this conserved mechanism. Assuming this is the case, the possible correlation between the temperature sensitivity of the resistance and the distinct N terminal structure of the three proteins in the current study is low. Transgenic complementation of susceptible Westonia plants with Sr6 was successful in transferring resistance against Pgt . Reduced resistance at high temperatures was also demonstrated in transgenic plants despite maintained expression of Sr6 , supporting a post-transcriptional or post-translational basis to temperature response. RNA analysis of the same transgenic plant transitioned between different temperatures may provide enhanced resolution of temperature dependent expression activity in future studies. The presence of the Sr6 sequence in all transgenic progeny was unexpected and may be related to multiple insertions of the transgene, some non-functional. However, this is yet to be experimentally determined. The observation of residual resistance a in number of T 1 plants at high temperature may be related to transgene copy number or genomic insertion site, leading to over or under expression caused by position effects 51 . Further expression analysis on these resistance-retaining plants is needed to uncover the true mechanism. Temperature has been shown to affect the relative abundance of transcript isoforms. Takabatake et al. 52 found that the tobacco ( Nicotiana glutinosa ) N gene, which encodes an NLR, generates two alternative transcripts: a complete form encoding the full protein, and a truncated form encoding a protein lacking most of the LRR; both transcripts accumulated at 20°C, a permissive temperature, but not at 30°C, a non-permissive temperature in tobacco mosaic virus (TMV)-inoculated leaves. However, the levels of the complete isoform were always higher just before resistance-related cell death. Like Sr6 , the N gene can be reversibly inactivated, and this phenomenon is maintained in heterologous transgenic systems such as tomato and N. benthamiana , indicating a conservation of signalling components 53 . NLRs are not the only class of R genes that exhibit temperature dependency. Yr36 , conferring partial resistance to wheat stripe rust (caused by P. striiformis f. sp. tritici ), is more effective at higher greenhouse temperatures (25–30⁰C) and encodes a kinase with a START lipid binding domain 54 . Fu et al. 54 found that Yr36 generates multiple non-functional transcripts with truncations, and the functional isoform encoding the complete protein was upregulated at higher temperatures, mirroring the enhanced resistance seen at such temperatures. Stripe rust infection did not have a significant effect on functional transcript abundance compared to that of temperature. There is growing evidence that alternative splicing is one of the ways in which plants respond to temperature, as found in Arabidopsis with LATE ELONGATED HYPOCOTYL ( LHY ) which displayed temperature-associated isoform switching 55 . Similarly, the expression of Sr21 was enhanced at higher temperatures, and was shown to encode 10 splice isoforms, albeit within the 3’UTR. However, three specific isoforms had increased abundance at 24⁰C compared to 16⁰C 24 . Sr13 is also more effective at higher temperatures (25⁰C), but unlike Sr21 , transcript abundance was not affected by temperature 23 . Based on the above, temperature-dependent resistance appears to be mediated primarily by transcriptional and post-transcriptional regulation. However, post-translational regulation has also been implicated; Zhu et al. 18 proposed temperature sensitivity was mediated by levels of inactive versus active protein conformations at varying temperatures, citing evidence of mutant R proteins that acquired heat stability, allowing them to remain in an active state at usually non-permissive temperatures. Additionally, little is known about the involvement of downstream signalling elements. In the case of Sr13 , only PR gene expression was affected by temperature and not Sr13 expression per se. Kiraly et al. 56 suggested that in the case of the tobacco N protein, the expression of downstream signalling components was also impaired at high temperatures. Clearly, the disease resistance pathway involves a myriad of regulatory and signalling components, some of which could be influenced to different degrees by temperature. For both Sr21 and Sr13 , upregulation of pathogenesis related (PR) genes was observed in resistant plants at higher temperatures only when inoculated with Pgt . PR genes were not upregulated in susceptible plants. This was further demonstrated by the results of this study, which showed similar upregulation of these genes at higher temperature. However, shifts in gene expression in response to temperature were less pronounced in Sr21- bearing lines. Overall, these results indicate the expression response of such PR genes is determined not by absolute temperature, but by activity of the Sr gene under permissive conditions. Wheat lines with temperature-sensitive Sr6 , effective at lower temperatures, displayed largely unique differentially expressed genes and pathways compared to wheat lines with Sr13 and Sr21 , which are both more effective at higher temperatures. PR genes associated with the SA pathway were upregulated in wheat lines with all three genes relative to the controls under low temperatures for Sr6 and high temperatures for Sr13 and Sr21 . The expression of these PR genes was much higher in plants with Sr6 compared to the other two genes. This is consistent with the strong resistance phenotype of Sr6 compared to the intermediate phenotype of Sr13 and Sr21 . Our study revealed unique pathways that are expressed in wheat lines with Sr6 compared to Sr13 and Sr21 at different temperatures. Differential gene expression analysis revealed unique genes that are expressed during the onset of resistance or susceptibility. Several genes upregulated at low temperature in LMPG- Sr6 were also upregulated either during susceptibility or at high temperature in Sr13 and Sr21 lines. To further explore the details of these host pathogen interactions and possible molecular changes, study of these genes at the protein level will be necessary. Similarly, genes associated with susceptibility require further study along with pathogen virulence factors. Our study highlights key factors to consider for temperature-dependent experiments on Sr gene expression: the effect of races, the frequency of alternative transcripts, overall expression, and regulation of PR genes and the currently neglected effect of pre-inoculation temperature. The cloning of Sr6 adds to the suite of temperature-sensitive, cloned R genes in wheat. Further work will help to decipher the possibly shared mechanisms of temperature sensitivity underlying these genes and to what advantage, if any, temperature sensitivity has in plant immunity, keeping in mind that temperature effects will vary between controlled experiments and in the field. Additionally, the cloning of Sr6 can expedite marker assisted screening and breeding programs in places where Sr6 is still effective. Methods Plant materials for Sr6 cloning Seven M 3 putative point mutations for Sr6 in Chinese Spring*5/Red Egyptian 2D (CS/RE 2D substitution line) were used in RenSeq analysis, namely, 3704-6, 3706-7, 3981-4, 4002-6, 4190-6, 5047-4, and 5140-5. F 3 lines derived from reciprocal crosses of CS × CS/RE 2D were used in genetic analysis and confirmation of a diagnostic marker developed from the gene candidate. Fifteen seedlings per line were screened with Pgt race 21 − 2,3,7 (University of Sydney Culture no. 216) and resistant plants had IT 0;. The Australian cultivar Westonia was used for transformation, and T 1 progeny were assessed for validation of the candidate gene. Mutant generation Seeds of two batches (1,200 and 1,500) of chromosome substitution line CS/RE 2D were treated by 0.5 or 0.6% ethyl methanesulfonate (EMS), respectively, as described in Zhang et al. 57 . Homozygous susceptible point mutants from seven different plants were used in subsequent R gene enrichment. MutRenSeq High molecular weight (HMW) DNA was prepared from leaves of non-infected seedlings of mutant and WT lines of CS/RE 2D using the phenol-chloroform method as described in Lagudah et al. 58 . DNA quality was assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and 0.8% agarose gels. Target R gene enrichment was performed using the Triticeae RenSeq Bait Library V1 ( https://github.com/steuernb/MutantHunter ) as described in Steuernagel et al. 59 . Subsequent steps of the pipeline were carried out as described in Hewitt et al. 60 using the MuTrigo package ( https://github.com/TC-Hewitt/MuTrigo ) for SNP calling and candidate search. Re-assembly using reads pooled from all samples (to increase coverage and enhance contiguity) was performed using the BioKanga package v4.4.2 ( https://github.com/csiro-crop-informatics/biokanga ) to trim raw reads with biokanga filter , assemble reads with biokanga assemb , and create scaffolds with biokanga scaffold using option -P 1600 , with all other parameters as default 61 . Confirmation of SNPs and domain prediction PCR amplification of coding regions was performed on HMW DNA of mutants and WT. Products were cloned using a TOPO™ XL-2 vector cloning kit (Invitrogen, Mulgrave, VIC, Australia). Plasmid DNA from at least four colonies per product was isolated using an ISOLATE II Plasmid Mini Kit (Bioline, Alexandria, NSW, Australia) and was then Sanger sequenced using primers listed in Supplementary Table 6. Coding sequences and translation were predicted using FGENESH ( http://www.softberry.com/berry.phtml ) and amino acid substitutions were confirmed on alignment to mRNA sequence using CodonCode Aligner v8.0 ( https://www.codoncode.com/aligner/ ). NB-LRR motif annotations were created using NLR-Parser ( https://github.com/steuernb/NLR-Parser ), and Zfn-BED domain prediction was performed using SMART ( http://smart.embl.de ) with added PFAM (v8) domains 62 . Sr6 transformation A 8,160 bp genomic sequence of Sr6 , including all introns, 2 kb upstream of 5′UTR and 1 kb downstream of the putative stop codon, encompassing the native promoter and terminator, was synthesized and cloned (Epoch Life Sciences, Missouri City, TX, USA) into Not I/ Sgs I-digested binary vector VecBarIII. Agrobacterium strain AGL1 was used to carry the construct and the Sr6 gene was introduced into cv. Westonia using the Agrobacterium-transformation protocol 63 and phosphinothricin as the selective agent. Two independent transgenic plants (T 0 ) carrying the Sr6 transgene were recovered as confirmed with both PCR markers Sr6STS1 and Sr6STS2 (Supplementary Table 6). Seed from both T 0 plants were harvested and sets of 25 T 1 seedlings were grown at low (18℃) and high (25℃) temperatures in growth cabinets along with WT and positive and negative controls. After two weeks, the WT, controls and T 1 plants were inoculated with Pgt race 21 − 0 (University of Sydney Culture no. 330). Phenotyping and photography of leaf samples were performed at 14 days post-inoculation. Planting, inoculation, and sample collection for RNAseq Near-isogenic wheat lines carrying Sr6 , Sr13 and Sr21 , along with recurrent parent LMPG-6 64 were planted in coarse vermiculite in plastic pots (5 x 5 cm). Twenty seeds of a given line were planted in each pot. The North American wheat stem rust differential set 12 was planted for each treatment. Temperature treatments were conducted under high (25 − 22℃) and low (18 − 15℃) regimes (15-hr day/9-hr night) in controlled-environment cabinets. Pathogen treatments included spraying with either Pgt urediniospores mixed with Soltrol® or only Soltrol as a mock-inoculated treatment. Ten-day-old wheat seedlings were inoculated with urediniospores of Pgt isolate 59KS19 (race MCCFC, avirulent to Sr6 , Sr13 , and Sr21 ) according to previously described conditions 65 . A total of 20 plants of each line were included in each temperature and pathogen treatment. The entire experiment was repeated three times with different planting and inoculation dates. RNAseq was performed for each genotype at each temperature and inoculation treatment at two time points: 24 and 72 hours after inoculation. A total of six primary leaves were collected for RNAseq at each timepoint, temperature, and inoculation treatment. Altogether 96 samples were collected and subjected to RNAseq. Primary leaf tissue was collected at 24 h and 72 h post inoculation for RNAseq and qPCR experiments. Young leaves were collected in 2 ml flat bottom Eppendorf tubes. All samples were flash frozen in liquid nitrogen and stored at -80℃ for successive experiments. Seedling infection types on unharvested primary leaves were observed and recorded 14 days post-inoculation 66 . Infection types (IT) were rated on a 0–4 scale with ‘0’ indicating no visible symptoms and ‘4’ being completely susceptible. IT greater than or equal to ‘3’ were considered susceptible and less than ‘3’ were considered resistant. IT were recorded on 5–6 seedlings of each control and experimental line in each treatment. RNA isolation, cDNA library construction, and sequencing The collected tissues were subjected to RNA extraction using Qiagen plant RNeasy kits. On-column DNase digestion (30 min) was performed to remove possible DNA contamination. RNA was eluted from each sample using 50 µl of elution buffer and quality-checked on a Nanodrop ND-1000 spectrophotometer and agarose gel electrophoresis. Pre-screened RNA samples were subjected to TrueSeq and RiboZero library preparation at the University of Minnesota Genomics Center and quality checked on Bioanalyzer chips (Agilent Technologies). Prepared libraries were first sequenced on a NovaSeq 6000 S1 flow cell to obtain 100 bp PE reads with a target of 35 million reads per sample followed by a second sequencing run on a NovaSeq 6000 S4 flow cell to obtain 150 PE reads with a target of 37 million reads per sample. The sequence reads were quality checked using FastQC and quality trimmed to 100 bp PE by removing barcodes and Illumina adapter sequences. An index of high-confidence protein coding sequences obtained from the IWGSC RefSeq annotation v1.1 was created with kallisto 0.46.1 67 . The transcript sequences were also aligned to this index using kallisto ( quant command) with 1,000 bootstraps of the PE reads to obtain alignment statistics. Alignment counts were further examined for differentially expressed genes (DEGs) in the R package edgeR 68 considering log fold change ≥ 2 and a false discovery rate (FDR) of 1 count per million (CPM) in at least three samples. Several DEGs were validated through qPCR. Genes unique to all three lines along with common genes with similar expression patterns in all three lines were selected for qPCR. RNA analysis and qPCR For qPCR, RNA isolated from leaf tissue at 0, 1 and 3 dpi were reverse transcribed to cDNA using protoScript® II Reverse Transcriptase (NEB). The reactions were carried out using probe-based IDT primers, 6FAM and IBFQ quencher, using 18S and GADPH as internal controls for gene expression analysis. TaqMan gene expression master mix (Applied Biosystems; Foster City, CA) and ABI 7300 (Applied Biosystems) were used for qPCR. The expression analysis was conducted using 2 −ΔΔC T to determine the relative quantification 69 . The fold expression of Sr lines was compared with the susceptible LMPG-6 lines at low and high temperature. A total of four upregulated genes from RNAseq were selected to confirm their relative expression. Additionally, pathogenesis-related (PR) resistance genes associated with the salicylic acid and jasmonic acid pathways were selected and tested (Supplementary Tables 5.1, 5.2). Six PR and four JA genes were tested to identify how the corresponding pathways were associated with resistance and various temperatures. For each near-isogenic line, the expression of the relevant resistance gene ( Sr6 , Sr13 , or Sr21 ) was tested at day 1 and day 3 at both low and high temperature treatments. RT-qPCR was carried out independently on transgenic Sr6 material using an alternative method. First, leaf tissues from each sample were frozen in liquid nitrogen or dry ice immediately after sampling; RNA was isolated using a RNeasy® Plant Mini Kit (QIAGEN, Chadstone Center, VIC, Australia) according to the manufacturer’s protocol, and used in first-strand DNA synthesis in 20 µL reactions using a Superscript® III reverse transcriptase kit (Life Technologies, Mulgrave, VIC, Australia). After the reverse transcript reaction, 3 µL of 10 ng/µL cDNA product was used for qPCR using a C1000 TouchTM thermocycler with the CFX96TM Real-Time System (Bio-Rad). qPCR conditions included an initial denaturation at 95°C for 3 min; 40 cycles of denaturation at 95°C for 10 s and annealing/elongation at 60°C for 30 s, followed by a melt step range of 65–95°C with increments of 0.5°C. The wheat housekeeping gene TaCON was used as the reference gene for each qRT-PCR experiment 70 . qPCR primers specific for Sr6 (forward: 5’-GTCAATAGCGCCGAGTGTAAG-3’ , reverse: 5’-GGTCTGATGGCTGAATTACTGG-3’ ) were used to measure relative gene expression using three technical replicates for each sample. δCq mean values were calculated and standard errors were determined. Gene expression values were log (base 2)-transformed. Boxplots of aggregated relative expression values for each temperature regime and disease phenotype were generated using geom_boxplot and geom_point functions of R package ggplot2 (v3.3.6). Welch’s two-tailed unpaired t-tests were performed using the t.test function from base R. RNA for rapid amplification of cDNA ends (RACE) was extracted from leaves of unchallenged, three-week-old seedlings of CS/RE 2D grown at ambient temperature (20–25⁰C) using the RNeasy Plant Mini Kit (Qiagen); 5’ and 3’ RACE was conducted using a SMARTer RACE 5’/3’ Kit (Clontech, Mountain View, CA, USA). RACE products were cloned using a TOPO™ XL-2 vector cloning kit (Invitrogen). Plasmids were isolated from 10 colonies per product using an ISOLATE II Plasmid Mini Kit (Bioline) and Sanger sequenced. Gene annotation and pathway analysis Pathway analysis and biological functions of differentially expressed genes were conducted via GeneOntology 71 , AgrigoV2 72 , and ShinyGo 73 . AgrigoV2 was used to identify the gene networks and pathways associated with the DEGs in different NILs at low or high temperature. GeneOntology software was used to categorize the DEGs into gene, family, protein, and species groups. The distribution of DEGs according to functional processes were represented in pie charts (Supplementary Figs. 10 and 11). ShinyGo was used to plot the DEGs across the chromosomes and calculate the statistical significance of various genomic regions 73 . Graphical enrichment analysis was completed using ShinyGo where query genes were mapped to the list of genes in particular pathways (Supplementary Figs. 12–13, 17–20). Fold enrichment was calculated by dividing the percentage of genes in the query list associated with a given pathway by the corresponding percentage of the genes in the background. FDR indicated the chances of fold enrichment and was calculated according to the Hypergeometric test 73 . Primer design and reference comparison Locus-specific primers were designed manually based on polymorphisms in alignments between Sr6 and high-scoring BLAST hits with Chinese Spring IWGSC RefSeq v1.0 (CSv1). DNA was isolated from leaves of F 3 lines using the method described in Ellis et al. 74 conducted on a Microlab NIMBUS™ liquid handling robot (Hamilton, Reno, NV). STS marker screens and gene amplification were carried out using primers listed in Supplementary Table 6. Products were checked by electrophoresis on 1% agarose gels. Marker Xwmc453 proximal to Sr6 32 was positioned on CSv1 chromosome 2D using BLAST v2.3 ( https://www.ncbi.nlm.nih.gov/books/NBK131777/ ) with the probe sequence retrieved from GrainGenes ( https://wheat.pw.usda.gov/GG3/ ). Homologous Sr6 sequences were identified from CSv1 and various reference assemblies of the wheat 10 + Genome Project ( http://www.10wheatgenomes.com/ ), listed in Adamski et al. 75 , using BLAST v2.9. Full protein sequences of Yr5 and Yr7 provided in confidence were aligned with Sr6 using Clustal Omega ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ). The transcript assembly of wheat cv. Avocet R used was the same as that described in Hewitt et al. 60 . NLR dendrogram construction NLR protein sequences with an N-terminal coiled-coil domain (CNL class) were taken from the NCBI database. Accession numbers are listed in Supplementary Table 7; 125 sequences were aligned using MUSCLE and a phylogenetic tree was generated using the UPGMA method in MEGA X ( www.megasoftware.net ). Distances were computed using the Poisson correction method and are in units of the number of amino acid substitutions per site. Protein structural modelling and analysis Full length protein structure, ATP and zinc ion binding regions in Fig. 1 B were predicted by AlphaFold3 via AlphaFold Server at https://golgi.sandbox.google.com/ 38 . Protein structures in Supplementary Figs. S14 and S15 were predicted by AlphaFold2 using ColabFold ( https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/beta/AlphaFold2_advanced.ipynb ) 76,77 . Structural and sequence analyses were carried out with PyMOL v2.5.5 and CLC Sequence Viewer v8. Weblogo diagram in Supplementary Fig. 16C was generated at https://weblogo.berkeley.edu/logo.cgi . Declarations Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Data availability The datasets and plant materials generated and analyzed in the current study are available from the corresponding authors. The data that support the findings of this study are openly available in NCBI, the mRNA sequence of Sr6 have been deposited at NCBI GenBank with accession number PP949235. Sequencing read data for MutRenSeq is deposited at DDBJ/EMBL/GenBank under BioProject PRJNA1127689. Other data are available within the paper and its Supplementary Information files. Source data are provided in this paper. Acknowledgements T.C.H., S.S., R.Mc., E.L., and P.Z. acknowledge the support from Grains Research and Development Corporation (GRDC), Australia. J.Z. acknowledges the support from the Australian Research Council (ARC) Early Career Industry Fellowship. M.N.R. acknowledges support from the USDA-ARS National Plant Disease Recovery System and a fellowship under the OECD Co-operative Research Programme: Biological Resource Management for Sustainable Agricultural Systems. T.C.H., J.Z., E.L., and P.Z. thank Dr. Chunhong Chen, CSIRO, for his excellent assistance. Author contributions P.Z., R.M., J.Z., S.S.: generation of mutants, disease phenotyping, and genotyping; T.H.: Sr6 mutant data analysis and candidate validation. 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Supplementary Files Supplementaryinformation.pdf SupplementaryTables25.xlsx Supplemental Tables 2-5 SupplementaryTables67.pdf Supplemental Tables 6-7 RS.pdf Report Summary EPC.pdf Editorial Policy checklist Cite Share Download PDF Status: Published Journal Publication published 28 May, 2025 Read the published version in Nature Communications → Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4674841","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":326362765,"identity":"2b006930-c926-439a-9598-e6d1b6be31c7","order_by":0,"name":"Peng 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Rouse","email":"","orcid":"https://orcid.org/0000-0001-7763-8203","institution":"USDA-ARS","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"","lastName":"Rouse","suffix":""}],"badges":[],"createdAt":"2024-07-02 14:10:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4674841/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4674841/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-60030-x","type":"published","date":"2025-05-28T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60386257,"identity":"fcafc7ad-0bd0-483b-8a41-f10f2a18a760","added_by":"auto","created_at":"2024-07-16 08:05:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":538952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSr6 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene (A) and SR6 protein (B) structures. A. \u003c/strong\u003e\u003cem\u003eSr6\u003c/em\u003e gene\u003cstrong\u003e \u003c/strong\u003estructure showing positions of EMS-induced SNPs identified in mutants.\u003cstrong\u003e \u003c/strong\u003eSNP positions are shown by red bars labelled with mutant number, altered amino acid positions are given. Shaded areas are UTRs. Domain encoding regions are indicated: CC, coiled-coil; NB-ARC, nucleotide binding (APAF-1, R proteins and CED-4); LRR, leucine rich repeat. \u003cstrong\u003eB\u003c/strong\u003e. Full length structure of SR6 protein, its zinc ion binding region and ATP binding region are predicted by AlphaFold3. The full length structure comprises three subdomains, the BED motif (light pink shaded) containing N terminal subdomain (yellow shaded), the NB-ARC domain (salmon shaded), and the LRR domain (green shaded).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4674841/v1/ba8853e7a235ac2c84f4a18b.png"},{"id":60386260,"identity":"8238dfdf-cd8f-4fd8-b2c6-58ca49e8a565","added_by":"auto","created_at":"2024-07-16 08:05:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":816473,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWestonia+\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSr6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e transgenic T\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e seedling leaves at 14 days post-inoculation with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePgt\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e race 21-0.\u003c/strong\u003e All numbered individuals were derived from the same T\u003csub\u003e0\u003c/sub\u003e parent (PC326-1) and grown at either 18℃ (A) or 25℃ (B). Only some representative leaves are shown. \u003cem\u003eSr6\u003c/em\u003e+ control: CS/RE 2D; \u003cem\u003eSr6\u003c/em\u003e- controls: Westonia, CS. Bottom labels indicate disease phenotype scored as either resistant (R), moderately resistant/moderately susceptible (M), moderate susceptible (MS), and susceptible (S). Relative gene expression (RE) values from qPCR results based on triplicate samples are listed. MS and S phenotypes have RE of 0.0.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4674841/v1/83ad17fda5e04ace047fd231.png"},{"id":60387208,"identity":"7cf7deff-c5ad-46da-b4f4-d6ad0177f1fd","added_by":"auto","created_at":"2024-07-16 08:21:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1656100,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSeedling responses to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePuccinia graminis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e f. sp. \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003etritici\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e race MCCFC under high and low temperature regimes for four wheat lines\u003c/strong\u003e: \u003cstrong\u003eA\u003c/strong\u003e: LMPG, \u003cstrong\u003eB\u003c/strong\u003e: LMPG-\u003cem\u003eSr6\u003c/em\u003e, \u003cstrong\u003eC\u003c/strong\u003e: LMPG-\u003cem\u003eSr13\u003c/em\u003e, and \u003cstrong\u003eD\u003c/strong\u003e: LMPG-\u003cem\u003eSr21\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4674841/v1/7be57f9e3b56381bfadad4cf.png"},{"id":60386265,"identity":"61369cc9-ff4d-4ae0-b84c-74ffacffbba1","added_by":"auto","created_at":"2024-07-16 08:05:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":51101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBoxplots representing qPCR relative expression (RE) values of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSr6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e transgene\u003c/strong\u003e from T\u003csub\u003e1\u003c/sub\u003e individuals grown at 18℃ (left panel) or 25℃ (right panel) grouped by resistant (R) or susceptible (S) disease phenotype 14 days post-inoculation with \u003cem\u003ePgt\u003c/em\u003e race 21-0. ** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4674841/v1/33c40228ce4e8ab4fa7729e8.png"},{"id":83593218,"identity":"6406b11f-201c-480b-8d82-840dd536881f","added_by":"auto","created_at":"2025-05-29 07:07:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4099214,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4674841/v1/8383c665-dae4-4183-86f4-052bbc0264fc.pdf"},{"id":60386757,"identity":"33ca7a5a-fbe2-455d-aed3-911c30b783a9","added_by":"auto","created_at":"2024-07-16 08:13:11","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6157617,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Supplementaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4674841/v1/b0b20e0b19915daec3d93850.pdf"},{"id":60386755,"identity":"bf1a0db9-65c8-4f00-9fee-75ae5ac0121d","added_by":"auto","created_at":"2024-07-16 08:13:11","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":146717,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Tables 2-5\u003c/p\u003e","description":"","filename":"SupplementaryTables25.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4674841/v1/c9a4df499f78fb6d026fbbad.xlsx"},{"id":60386266,"identity":"cc689cad-ffc9-408f-be07-8c5d71b9761a","added_by":"auto","created_at":"2024-07-16 08:05:14","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":694586,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Tables 6-7\u003c/p\u003e","description":"","filename":"SupplementaryTables67.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4674841/v1/82bd6efbbfdc93158afa695f.pdf"},{"id":60386264,"identity":"6e3946b7-f686-4b77-8ea7-6d1745c8e49c","added_by":"auto","created_at":"2024-07-16 08:05:11","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1522805,"visible":true,"origin":"","legend":"Report Summary","description":"","filename":"RS.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4674841/v1/ee17d9cd8af59160469efacc.pdf"},{"id":60386262,"identity":"9e560c52-bdc0-4aef-a1d1-baa948730225","added_by":"auto","created_at":"2024-07-16 08:05:11","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1509329,"visible":true,"origin":"","legend":"Editorial Policy checklist","description":"","filename":"EPC.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4674841/v1/593f7e78b85e5d99be802768.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Divergent molecular pathways govern temperature-dependent wheat stem rust resistance genes Sr6, Sr13 and Sr21","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWheat stem rust, caused by the fungal pathogen \u003cem\u003ePuccinia graminis\u003c/em\u003e f. sp. \u003cem\u003etritici\u003c/em\u003e (\u003cem\u003ePgt\u003c/em\u003e), poses a significant threat to global wheat production. Virulent strains have overcome widely deployed disease resistance genes, including \u003cem\u003eSr6\u003c/em\u003e\u003csup\u003e1\u003c/sup\u003e. This gene, once widely effective, is now widespread and found in about 13% of global spring wheat varieties\u003csup\u003e2\u003c/sup\u003e. Stem rust epidemics have historically caused severe damage during warm weather, affecting continental-scale wheat production\u003csup\u003e3,4\u003c/sup\u003e. In the United States, stem rust outbreaks from 1919 to 1954 led to substantial statewide losses\u003csup\u003e5\u003c/sup\u003e often causing up to 50% yield loss\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe release of the \u003cem\u003eSr6\u003c/em\u003e-bearing cultivar Selkirk was particularly successful in combatting stem rust epidemics that swept across North America from the 1920s to 1960s. The first widely grown Australian stem rust resistant cultivar was Eureka carrying \u003cem\u003eSr6\u003c/em\u003e and released in 1938. However, as early as in 1942, virulent \u003cem\u003ePgt\u003c/em\u003e races were identified, and the cultivar went through a series of recurrent epidemics before it was removed from cultivation after the mid-1960s\u003csup\u003e6\u003c/sup\u003e. Nevertheless, \u003cem\u003eSr6\u003c/em\u003e still provides resistance in some parts of North America\u003csup\u003e7,8\u003c/sup\u003e and possibly India\u003csup\u003e9\u003c/sup\u003e, particularly when combined with other genes such as \u003cem\u003eSr57\u003c/em\u003e (\u003cem\u003eLr34\u003c/em\u003e) \u003csup\u003e10\u003c/sup\u003e or \u003cem\u003eSr2\u003c/em\u003e (as was the case with Selkirk).\u003c/p\u003e \u003cp\u003eThe emergence of \u003cem\u003ePgt\u003c/em\u003e races TTKSK (commonly referred to as Ug99) and TKTTF (\u0026lsquo;Digalu\u0026rsquo; race) in East Africa was particularly alarming. These races were virulent to widely deployed stem rust resistance genes, rendering 80\u0026ndash;90% of global wheat varieties susceptible\u003csup\u003e11\u0026ndash;15\u003c/sup\u003e. Spread across multiple regions, the Ug99 and Digalu races and variants overcame resistance genes such as \u003cem\u003eSr24\u003c/em\u003e, \u003cem\u003eSr36\u003c/em\u003e, and \u003cem\u003eSrTmp\u003c/em\u003e\u003csup\u003e11\u0026ndash;13,16\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePlants employ basal and resistance (R)-mediated defense responses to infection by microbial pathogens\u003csup\u003e17\u003c/sup\u003e. Both responses are influenced by heat stress\u003csup\u003e18\u003c/sup\u003e, highlighting the adaptability of the plant defense system to varying temperatures. Pathogen-Associated Molecular Patterns (PAMPs) activate PAMP-triggered immunity (PTI) upon detection. Host-adapted pathogens manipulate PTI using effector proteins, which can be recognized by plant intracellular receptors, leading to Effector-Triggered Immunity (ETI). ETI, induced by host R genes, results in hypersensitive responses or programmed cell death\u003csup\u003e18\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDurable disease resistance is essential to safeguard wheat from ever-changing rust pathogens\u003csup\u003e19\u003c/sup\u003e. Stem rust resistance is often placed in two categories: Adult plant resistance (APR), conferred by multi-pathogen resistance genes such as \u003cem\u003eSr2, Sr57\u003c/em\u003e/\u003cem\u003eLr34 Sr58\u003c/em\u003e/\u003cem\u003eL46\u003c/em\u003e and \u003cem\u003eSr55\u003c/em\u003e/\u003cem\u003eLr67\u003c/em\u003e (and other designations) that show effectiveness at later growth stages, and all-stage resistance (ASR), effective from the seedling stage. Cloned ASR genes for stem rust include \u003cem\u003eSr22, Sr26, Sr33, Sr35, Sr45\u003c/em\u003e, \u003cem\u003eSr50\u003c/em\u003e, \u003cem\u003eSr61\u003c/em\u003e, and \u003cem\u003eSr62\u003c/em\u003e as well as the temperature sensitive \u003cem\u003eSr13\u003c/em\u003e and \u003cem\u003eSr21\u003c/em\u003e\u003csup\u003e\u003cem\u003e20\u0026ndash;26\u003c/em\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThese \u003cem\u003eR\u003c/em\u003e genes typically encode coiled coil-nucleotide binding leucine-rich repeat (CC-NBS-LRR or NLR) proteins or protein kinases, such as \u003cem\u003eSr60\u003c/em\u003e\u003csup\u003e\u003cem\u003e27\u003c/em\u003e\u003c/sup\u003e. Notably, \u003cem\u003eSr13\u003c/em\u003e and \u003cem\u003eSr21\u003c/em\u003e are more effective at higher temperatures\u003csup\u003e1,11,23,24,28\u003c/sup\u003e, in contrast to \u003cem\u003eSr6\u003c/em\u003e which is more effective below 20\u0026deg;C and ineffective above 24\u0026ndash;27\u0026deg;C\u003csup\u003e29,30\u003c/sup\u003e. Similarly, stem rust resistance genes \u003cem\u003eSr10, Sr15\u003c/em\u003e, and \u003cem\u003eSr17\u003c/em\u003e are less effective at higher temperatures\u003csup\u003e24\u003c/sup\u003e. Plants susceptible at a non-permissive temperature regained resistance once the temperature returned to a permissive level\u003csup\u003e31\u003c/sup\u003e. Light conditions also impact the resistance response\u003csup\u003e32\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhereas \u003cem\u003eSr13\u003c/em\u003e and \u003cem\u003eSr21\u003c/em\u003e were previously cloned\u003csup\u003e23,24\u003c/sup\u003e, here we report the cloning of \u003cem\u003eSr6\u003c/em\u003e using mutagenesis and R gene enrichment and sequencing (MutRenSeq) and show that it is an NLR integrated with a non-canonical zinc finger BED domain. Stable transformation confirmed \u003cem\u003eSr6\u003c/em\u003e identity which induced a characteristic temperature-sensitive resistance phenotype in transgenic plants challenged with an \u003cem\u003eSr6-\u003c/em\u003eavirulent \u003cem\u003ePgt\u003c/em\u003e race. RNA sequence analysis showed that gene expression was affected by temperature. Differential gene expression analysis on near-isogenic wheat lines carrying either \u003cem\u003eSr6\u003c/em\u003e, \u003cem\u003eSr13\u003c/em\u003e or \u003cem\u003eSr21\u003c/em\u003e elucidated varied defence pathways in response to different temperatures.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eSr6\u003c/b\u003e \u003cb\u003ecandidate isolated by MutRenSeq and verified in recombinant inbred lines\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo clone \u003cem\u003eSr6\u003c/em\u003e, we identified susceptible EMS-generated mutants from the substitution line Chinese Spring*5/Red Egyptian 2D (CS/RE 2D) produced by Sears et al. \u003csup\u003e33\u003c/sup\u003e. Seven independent mutants together with wild-type CS/RE 2D were processed using the MutRenSeq pipeline. Captured sequencing reads from these seven mutants and the wild type (WT) were aligned to a \u003cem\u003ede novo\u003c/em\u003e reference assembly of the WT reads. One contig (#733836) of 2,842 bp containing a SNP was identified in three of the seven mutants and possessed NB-ARC and LRR motifs but no CC motif. A second contig (#737511) of 2,218 bp containing a SNP in a different three mutants was identified and possessed only LRR motifs. The last\u0026thinsp;~\u0026thinsp;600 bp of contig #733836 had homology to the first\u0026thinsp;~\u0026thinsp;600 bp of contig #737511 suggesting they overlapped but were not joined during assembly possibly due to low coverage and/or ambiguity of reads covering the bridging region. To confirm that these sequences were physically joined, a second assembly was generated based on a combined pool of reads from all the mutants and WT. The second assembly produced a scaffold (#2265) that represented a joining of the two contigs in six of the seven mutants having SNPs (Supplementary Fig.\u0026nbsp;1A).\u003c/p\u003e \u003cp\u003eNo SNP was detected in mutant 3981-4, but since scaffold #2265 did not contain any CC domain-encoding motifs, mutant 3981-4 was predicted to harbour a mutation in a potentially missing upstream sequence. Scaffold #2265 was aligned to the Chinese Spring RefSeq v1.0 (CSv1) reference assembly\u003csup\u003e34\u003c/sup\u003e using BLAST, with the top hit (~\u0026thinsp;91%) to chromosome 2D approximately 5.1 Mbp from the \u003cem\u003eSr6\u003c/em\u003e-proximal marker \u003cem\u003eXwmc453\u003c/em\u003e identified by Tsilo et al. \u003csup\u003e\u003cem\u003e32\u003c/em\u003e\u003c/sup\u003e. The matching sequence overlapped with high-confidence annotated gene \u003cem\u003eTraesCS2D02G111500\u003c/em\u003e, encoding a disease resistance protein. A dominant PCR marker \u003cem\u003eSr6STS1\u003c/em\u003e designed based on the sequence of scaffold #2265 showed specificity to \u003cem\u003eSr6\u003c/em\u003e-carrying lines CS/RE 2D and Manitou (Supplementary Fig.\u0026nbsp;2A).\u003c/p\u003e \u003cp\u003eAdditionally, a PCR product spanning almost the full length of scaffold #2265 confirmed linkage of the two initially discovered contigs (Supplementary Fig.\u0026nbsp;2B) and was amplified from the six mutants with SNPs. Sanger sequencing confirmed presence of the SNP mutations. Each of the six mutants contained a nonsynonymous SNP, with five resulting in missense mutations and one in a nonsense mutation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). 5\u0026rsquo;RACE (rapid amplification of cDNA ends) from WT RNA revealed the presence of an additional 5\u0026rsquo; sequence that encoded both CC and zinc finger BED domains. Primers were designed to amplify this additional 5\u0026rsquo; region from DNA of mutant 3981-4 (Supplementary Fig.\u0026nbsp;2C), from which a mutation had yet to be identified. Sanger sequencing revealed the presence of a G-to-A SNP (Supplementary Fig.\u0026nbsp;1B) in the 5\u0026rsquo;UTR, 20 bp upstream of the first start codon. Although this mutation does not alter the coding sequence, mutations in the 5\u0026rsquo;UTR are known to impact translation\u003csup\u003e35,36\u003c/sup\u003e. Overall, point mutations were confirmed in the candidate sequence for all seven mutants (Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further verify the candidate as \u003cem\u003eSr6\u003c/em\u003e, a population of 197 F\u003csub\u003e3\u003c/sub\u003e lines from a reciprocal cross of CS and CS/RE 2D segregating for \u003cem\u003eSr6\u003c/em\u003e were screened for response to \u003cem\u003ePgt\u003c/em\u003e race 21\u0026thinsp;\u0026minus;\u0026thinsp;2,3,7. The dominant STS marker \u003cem\u003eSr6STS1\u003c/em\u003e, based on the candidate sequence, was present in all homozygous resistant and segregating lines, and absent in homozygous susceptible lines.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFull gene structure of\u003c/b\u003e \u003cb\u003eSr6\u003c/b\u003e \u003cb\u003eobtained from whole genome sequenced wheat accessions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eSr6\u003c/em\u003e candidate sequence compared against published wheat genome assemblies\u003csup\u003e37\u003c/sup\u003e had a 100% match on chromosome 2D of the genome assembly of cv. Landmark (Supplementary Fig.\u0026nbsp;3). The full upstream and downstream sequences from Landmark presented an opportunity to use the native regulatory elements for transgenic experiments with \u003cem\u003eSr6\u003c/em\u003e. However, the probable promoter region, less than 2 kb upstream of the putative start of codon, contained a large gap in sequence denoted by Ns (unknown bases). Fortunately, the \u003cem\u003eSr6\u003c/em\u003e sequence was also found in an assembled scaffold of cv. Claire harbouring a gapless upstream sequence. The sequence from Claire was used to close the gap present in the Landmark sequence (Supplementary Fig.\u0026nbsp;4A). Additionally, a scaffold from cv. Robigus, which contained a non-identical coding sequence, did have an identical upstream region to Claire, indicating the sequence overlapping the gap was conserved.\u003c/p\u003e \u003cp\u003eA transcript was identified from 5\u0026rsquo; and 3\u0026rsquo; RACE amplification having an intron/exon structure showing conservation with the CSv1 homologue \u003cem\u003eTraesCS2D02G111500.\u003c/em\u003e Additionally, a complete transcript retrieved from the \u003cem\u003ede novo\u003c/em\u003e transcriptome assembly of Avocet R supported conservation of the intron/exon structure at the 5\u0026rsquo; end (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSr6\u003c/b\u003e \u003cb\u003eis diverged from known wheat NLRs but contains a conserved BED domain\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eSr6\u003c/em\u003e candidate was aligned to 16 reference genomes, of which, only Landmark and Claire contained a matching sequence. However, the ~\u0026thinsp;165 bp BED domain encoding region (exon 2) appears conserved, as seen in the alignment with the Avocet R ortholog (Supplementary Fig.\u0026nbsp;3). The BED domain sequence was also identical in the chromosome 2D homologs in ArinaLrFor, Jagger, Julius, Lancer, Norin 61 and Robigus. The BED domains of the 2D homologs in CSv1 and Weebill 1, as well as the chromosome 2B homoeolog in Zavitan (tetraploid) had eight SNPs with \u003cem\u003eSr6\u003c/em\u003e. The 2D homologs in Mace, SY Mattis and spelt accession PI190962 had 16 SNPs.\u003c/p\u003e \u003cp\u003eThe full length protein of SR6, its zinc ion binding region and ATP binding region were predicted by AlpahFold3\u003csup\u003e38\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The BED domain of \u003cem\u003eSr6\u003c/em\u003e was also compared to those of stripe rust R genes \u003cem\u003eYr5\u003c/em\u003e and \u003cem\u003eYr7\u003c/em\u003e, the only cloned wheat R genes thus far known to contain a BED domain\u003csup\u003e39\u003c/sup\u003e. \u003cem\u003eYr5\u003c/em\u003e and \u003cem\u003eYr7\u003c/em\u003e have the same BED-NB-LRR configuration as \u003cem\u003eSr6\u003c/em\u003e and have a similar intron/exon arrangement in that each of the CC, BED and NB-LRR domains reside in its own exon. Whereas the protein sequence of the \u003cem\u003eSr6\u003c/em\u003e candidate is quite differentiated from that of \u003cem\u003eYr5/Yr7\u003c/em\u003e, 12 residues in the BED domain appear conserved (Supplementary Fig.\u0026nbsp;4B).\u003c/p\u003e \u003cp\u003eThe translated sequence of the \u003cem\u003eSr6\u003c/em\u003e candidate was compared against the NCBI protein database but produced no significant hits (\u0026lt;\u0026thinsp;80% sequence identity). Comparison of the protein sequence of the candidate against a panel of known CNL class NLRs did not indicate any close relationship although it did cluster with other wheat R genes including \u003cem\u003eSr21\u003c/em\u003e, \u003cem\u003eYr5\u003c/em\u003e and \u003cem\u003eYr7\u003c/em\u003e (Supplementary Fig.\u0026nbsp;5). A commonality between these three genes and \u003cem\u003eSr6\u003c/em\u003e is that they reside on group 2 chromosomes, but they are not located at orthologous positions as \u003cem\u003eSr6\u003c/em\u003e is in the 2DS arm whereas \u003cem\u003eSr21\u003c/em\u003e and \u003cem\u003eYr5/Yr7\u003c/em\u003e are in the 2AL and 2BL arms, respectively\u003csup\u003e24,39\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTransgenic complementation with the\u003c/b\u003e \u003cb\u003eSr6\u003c/b\u003e \u003cb\u003ecandidate confers temperature-sensitive resistance segregating in T1 generation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn addition to lines CS/RE 2D and Manitou, which are known to have \u003cem\u003eSr6\u003c/em\u003e\u003csup\u003e1\u003c/sup\u003e, several transformable varieties were tested with marker \u003cem\u003eSr6STS1\u003c/em\u003e to screen for an appropriate transgene host. Wheat varieties Fielder and Yitpi were positive whereas Westonia and durum cultivar Stewart were negative (data not shown). Furthermore, phenotyping the seed stocks of Fielder maintained at CSIRO Canberra identified low infection types (ITs) with \u003cem\u003eSr6\u003c/em\u003e-avirulent \u003cem\u003ePgt\u003c/em\u003e races 21\u0026thinsp;\u0026minus;\u0026thinsp;0 and 21\u0026thinsp;\u0026minus;\u0026thinsp;2,3,7, and high IT with \u003cem\u003eSr6\u003c/em\u003e-virulent race 21\u0026thinsp;\u0026minus;\u0026thinsp;1,2,3,5,6,(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) (University of Sydney Culture no. 50); Westonia had high ITs with all three races even though it was previously reported to carry \u003cem\u003eSr6\u003c/em\u003e\u003csup\u003e40\u003c/sup\u003e. Consequently, the CSIRO stock of Westonia was chosen as the susceptible host for transgenic complementation.\u003c/p\u003e \u003cp\u003eAgrobacterium transformation of embryos of Westonia with the \u003cem\u003eSr6\u003c/em\u003e candidate gene, including native regulatory elements, yielded two successful T\u003csub\u003e0\u003c/sub\u003e transformants. The T\u003csub\u003e1\u003c/sub\u003e progeny were grown at either low (18℃) or high (25℃) temperatures and were infected with \u003cem\u003ePgt\u003c/em\u003e race 21\u0026thinsp;\u0026minus;\u0026thinsp;0 isolate at two weeks old. The plants were scored for their resistance responses at 14 days post-inoculation (dpi) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Fig.\u0026nbsp;6). At low temperature, there was clear distinction between resistant (n\u0026thinsp;=\u0026thinsp;39) and susceptible (n\u0026thinsp;=\u0026thinsp;8) individuals across both parental batches (~\u0026thinsp;3:1 segregation overall, χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;1.60, p\u0026thinsp;=\u0026thinsp;0.207) where resistance was observed as small necrotic lesions or small uredinia surrounded by necrosis. At high temperature, the infection types were much higher overall and there were 13 susceptible and 35 resistant individuals (~\u0026thinsp;3:1 segregation overall, χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.11, p\u0026thinsp;=\u0026thinsp;0.739). The presence of resistant T\u003csub\u003e1\u003c/sub\u003e plants confirmed resistance activity of the transgene and its identity as \u003cem\u003eSr6\u003c/em\u003e. The higher ratio of susceptible plants at high temperature comports with expected loss of resistance seen with endogenous \u003cem\u003eSr6\u003c/em\u003e at high temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and suggests temperature sensitivity is mediated by the \u003cem\u003eSr6\u003c/em\u003e gene itself. Apparently 25℃ was not sufficient to achieve a complete knockout of \u003cem\u003eSr6\u003c/em\u003e with the genetic material and environmental conditions of the present experiment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAll T\u003csub\u003e1\u003c/sub\u003e individuals were assayed with the \u003cem\u003eSr6\u003c/em\u003e STS marker to check for co-segregation with resistance. Unexpectedly, all individuals were positive after repeated tests, suggesting one or more non-functional copies of the transgene integrated into the genome of all individuals. Position effect variegation of transgene expression among individuals may have led to segregation of resistant and susceptible phenotypes. \u003cem\u003eSr6\u003c/em\u003e expression was measured by qPCR and the relative expression (RE) was calculated for each individual and distributions of RE values from resistant and susceptible groups were compared between high and low temperature conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The difference between low-temperature resistant and high-temperature resistant groups was not significant (p\u0026thinsp;=\u0026thinsp;0.2883 Welch\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test), indicating temperature generally had minimal impact on expression. Whereas resistant plants had a wide range of RE values at low temperature, all susceptible plants showed little to no expression. In contrast, a considerable number of \u0026lsquo;susceptible\u0026rsquo; [moderately resistant/moderately susceptible (MRMS) and MS] plants (n\u0026thinsp;=\u0026thinsp;10) maintained relatively high levels of expression at high temperature, suggesting that suppression of resistance in these plants was not solely modulated by expression. The difference between low-temperature susceptible and high-temperature susceptible was significant (p\u0026thinsp;=\u0026thinsp;0.0019) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Unsurprisingly, the expression difference between resistant and susceptible groups was very high (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) at both temperatures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSr\u003c/b\u003e \u003cb\u003egene expression tracks with resistance response at different temperatures\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe NILs LMPG-\u003cem\u003eSr6\u003c/em\u003e, LMPG-\u003cem\u003eSr13\u003c/em\u003e and LMPG-Sr\u003cem\u003e21\u003c/em\u003e were inoculated with \u003cem\u003ePgt\u003c/em\u003e race MCCFC (isolate no 59KS19) and sampled at different timepoints. The \u003cem\u003eSr6\u003c/em\u003e NIL was highly resistant (IT \u0026lsquo;0\u0026rsquo;) at low temperature (18℃) but susceptible (IT \u0026lsquo;3\u0026thinsp;+\u0026thinsp;C\u0026rsquo;) at high temperature (25℃) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). \u003cem\u003eSr13\u003c/em\u003e and \u003cem\u003eSr21\u003c/em\u003e showed the opposite pattern where the corresponding lines were more resistant at the higher temperature (IT \u0026lsquo;2\u0026rsquo; and \u0026lsquo;2-\u0026rsquo;, respectively) compared to the low temperature (IT \u0026lsquo;2\u0026thinsp;+\u0026thinsp;3\u0026rsquo; and \u0026lsquo;2+\u0026rsquo;, respectively) (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eSr6\u003c/em\u003e was upregulated at low temperature (Supplementary Fig.\u0026nbsp;7.1). Specifically, the level of \u003cem\u003eSr6\u003c/em\u003e expression was higher at day 3 at low temperature but was lower at high temperature. At low temperature, \u003cem\u003eSr13\u003c/em\u003e was upregulated at day 1 but downregulated at day 3, whereas it was downregulated at day 1 and upregulated at day 3 at high temperature. \u003cem\u003eSr21\u003c/em\u003e was upregulated on both day 1 and day 3 under both low and high temperature conditions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiotic stress-related genes show differential expression between\u003c/b\u003e \u003cb\u003eSr\u003c/b\u003e \u003cb\u003elines at high and low temperatures\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUnder conditions leading to more resistant responses (high temperature for \u003cem\u003eSr13\u003c/em\u003e and \u003cem\u003eSr21\u003c/em\u003e; low temperature for \u003cem\u003eSr6\u003c/em\u003e), six pathogenesis related (PR) and four jasmonic acid (JA) pathway genes were tested by qPCR for gene expression (Supplementary Figs.\u0026nbsp;7.2\u0026ndash;7.4, Supplementary Tables\u0026nbsp;5.1, 5.2). All were upregulated in \u003cem\u003eSr6\u003c/em\u003e lines under low temperature, except \u003cem\u003ePR4\u003c/em\u003e which was downregulated on day 3. \u003cem\u003ePR1\u003c/em\u003e and \u003cem\u003ePR9\u003c/em\u003e were highly expressed at day 3 in \u003cem\u003eSr6\u003c/em\u003e lines. Most of the PR genes were upregulated in \u003cem\u003eSr13\u003c/em\u003e lines under high temperature except \u003cem\u003ePR3\u003c/em\u003e, \u003cem\u003ePR4\u003c/em\u003e and \u003cem\u003ePR5\u003c/em\u003e which were downregulated at day 3 (Supplementary Fig.\u0026nbsp;7.3A). In \u003cem\u003eSr21\u003c/em\u003e lines under high temperature, \u003cem\u003ePR3\u003c/em\u003e and \u003cem\u003ePR4\u003c/em\u003e were downregulated at day 1 whereas \u003cem\u003ePR1\u003c/em\u003e, \u003cem\u003ePR2\u003c/em\u003e, \u003cem\u003ePR5\u003c/em\u003e and \u003cem\u003ePR9\u003c/em\u003e were upregulated (Supplementary Fig.\u0026nbsp;7.4A). All PR genes were downregulated in \u003cem\u003eSr21\u003c/em\u003e lines at day 3. Expression of PR genes relates to the salicylic acid (SA) pathway and is known to be associated with resistance against pathogen attack\u003csup\u003e23\u003c/sup\u003e. Similarly, relative expression of several components of the jasmonic acid signalling were tested under high and low temperatures. Among the four tested JA related genes in \u003cem\u003eSr6\u003c/em\u003e lines, three were downregulated under low temperature except \u003cem\u003eJA3\u003c/em\u003e, which was upregulated at day 1 (Supplementary Fig.\u0026nbsp;7.2B). In \u003cem\u003eSr13\u003c/em\u003e lines, \u003cem\u003eJA1\u003c/em\u003e and \u003cem\u003eJA3\u003c/em\u003e were upregulated under high temperature at day 1, but all JA genes were downregulated in \u003cem\u003eSr13\u003c/em\u003e lines on all other conditions (Supplementary Fig.\u0026nbsp;7.3B). In \u003cem\u003eSr21\u003c/em\u003e lines, \u003cem\u003eJA\u003c/em\u003e related genes were downregulated (Supplementary Fig.\u0026nbsp;7.4B)\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNAseq analysis shows divergence in broader expression patterns between\u003c/b\u003e \u003cb\u003eSr\u003c/b\u003e \u003cb\u003elines\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDifferential expression of most genes (35,000\u0026ndash;36,000) was non-significant using our analysis criteria. RNAseq analysis indicated that 57 to 872 genes were upregulated in the NILs compared to parental LMPG at both low and high temperatures (Supplementary Fig.\u0026nbsp;8). Eight genes uniquely expressed in all three lines, i.e. four upregulated genes; Aquaporin PIP1mRNA (PIP1), Oxygen-evolving \u003cem\u003eenhancer\u003c/em\u003e protein-2 (OEE-2), Chlorophyll binding protein (CBP) and Alpha-amylase/trypsin inhibitor-like protein (AAMY) at day 3 were validated through qPCR analysis (Supplementary Table\u0026nbsp;5.3). The qPCR results validated the results obtained from RNAseq analysis (Supplementary Fig.\u0026nbsp;7.5). \u003cem\u003eOEE-2\u003c/em\u003e and \u003cem\u003eCBP\u003c/em\u003e were upregulated in LMPG-\u003cem\u003eSr6\u003c/em\u003e under low temperature but were downregulated under high temperature. \u003cem\u003ePIP1\u003c/em\u003e was downregulated under both low and high temperatures. \u003cem\u003eOEE-2\u003c/em\u003e was downregulated in LMPG-\u003cem\u003eSr13\u003c/em\u003e under high and low temperature whereas it was upregulated under high temperature in LMPG-\u003cem\u003eSr21. PIP1\u003c/em\u003e was upregulated in LMPG-\u003cem\u003eSr13\u003c/em\u003e and LMPG-\u003cem\u003eSr21\u003c/em\u003e and downregulated in LMPG-\u003cem\u003eSr6\u003c/em\u003e. Additionally, \u003cem\u003eAAMY\u003c/em\u003e was upregulated under both high and low temperatures in LMPG-\u003cem\u003eSr21\u003c/em\u003e. \u003cem\u003eCBP\u003c/em\u003e was downregulated under high temperature but upregulated under low temperature in LMPG-\u003cem\u003eSr21\u003c/em\u003e as in LMPG-\u003cem\u003eSr6\u003c/em\u003e. This indicated that \u003cem\u003eCBP\u003c/em\u003e showed a similar expression pattern for both a low- and a high-temperature-sensitive \u003cem\u003eSr\u003c/em\u003e gene.\u003c/p\u003e \u003cp\u003eMost DEGs in LMPG-\u003cem\u003eSr13\u003c/em\u003e and LMPG-\u003cem\u003eSr21\u003c/em\u003e followed a similar expression pattern that differed from LMPG-\u003cem\u003eSr6\u003c/em\u003e. The number of DEGs for a particular NIL under a certain temperature ranged from 26 to 553 (Supplementary Fig.\u0026nbsp;9). Most of the transcripts upregulated in LMPG-\u003cem\u003eSr6\u003c/em\u003e under low temperature (CR) were distributed across the genome, but with a higher number of genes mapped to chromosomes 2B and 3B on both day 1 and day 3 (Supplementary Fig.\u0026nbsp;10). Similarly, DEGs mapped to most chromosomes in LMPG-\u003cem\u003eSr13\u003c/em\u003e and LMPG-\u003cem\u003eSr21\u003c/em\u003e, with some variation where reads tended to map frequently to chromosomes 1A, 2B, and homoeologous group 7 members.\u003c/p\u003e \u003cp\u003eGene ontology (GO) analyses were conducted using GeneOntology and ShinyGo (Supplementary Figs.\u0026nbsp;10\u0026ndash;13, 17\u0026ndash;20; Supplementary Tables\u0026nbsp;2\u0026ndash;4). Enrichment analysis in \u003cem\u003eSr6\u003c/em\u003e lines demonstrated that genes related to defence response to fungal infection, response to biotic stimulus, and various pathogen resistance proteins were expressed under low temperature. These pathways corresponded with the strong upregulation of PR genes for \u003cem\u003eSr6\u003c/em\u003e at low temperature. On the contrary, significant pathways were not observed for LMPG-\u003cem\u003eSr13\u003c/em\u003e under low temperature on day 1. However, when at the high temperature where \u003cem\u003eSr13\u003c/em\u003e is most effective, numerous pathways including ribosomal small subunit assembly, carton utilization, and photosynthesis were upregulated. Upregulated genes included OEE-2 and Cytochrome C. For LMPG-\u003cem\u003eSr21\u003c/em\u003e proton transmembrane transport was upregulated on day 1 at both high and low temperatures. Numerous differences in upregulated pathways were observed at day 3 where various biosynthetic and metabolic processes were upregulated at high temperature, but not low temperature. Genes upregulated on day 3 included kinase proteins, an ABC transporter, OEE-2, and several membrane trafficking proteins.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eProtein structural comparison and analysis\u003c/h2\u003e \u003cp\u003eTo further investigate any potential association between temperature-sensitive resistance exhibited by SR6, SR13, and SR21, we performed in-silico protein structure modelling using AlphaFold3\u003csup\u003e38\u003c/sup\u003e. Full-length protein structure prediction of all three proteins clearly displayed three subdomains: an N terminal domain, an NB-ARC domain, and an LRR domain with overall high confidence (Supplementary Figs.\u0026nbsp;14A, 15). Although all three full length structures are well conserved in protein subdomain composition, the N terminal domains of SR6 and SR21 are significantly different from SR13 that contains a four helix bundle to comprise a coiled coil structure (Supplementary Fig.\u0026nbsp;14B), whereas the N terminal subdomain of SR6 formed by four helices with a BED domain motif insert between a3 and a4, the N terminal subdomain of SR21 appears to form an extra helix in addition to the four helices coiled coil bundle (Supplementary Fig.\u0026nbsp;14C). Nevertheless, despite all these distinctive N terminal structures, all three proteins seem to have a highly conserved first three helices bundle and noticeably the a3 helix is always the helix that is physically most adjacent to the LRR domain (Supplementary Fig.\u0026nbsp;14C). We further compared the zinc finger BED motif from SR6 with other zinc finger BED motifs from other BED domain-containing resistance proteins. All zinc finger BED structures from SR6, YR5a, RPH15, and XA1 highly resembled each other with pairwise Root-Mean-Square Deviation (RMSD) value less than 1 (Supplementary Fig.\u0026nbsp;16A), despite the rather low sequence similarity (Supplementary Fig.\u0026nbsp;16B). The WebLogo diagram unsurprisingly displayed a high conservation only for cysteines and histidines, which are predicted to bind to the zinc ions (Zn\u003csup\u003e2+\u003c/sup\u003e) and resemble a \u0026ldquo;finger\u0026rdquo; shape (Supplementary Fig.\u0026nbsp;16C). Overall, we did not identify any obvious correlation between the resistance temperature sensitivity and the protein structure composition of SR6, SR13, and SR21.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMourad et al.\u003csup\u003e8\u003c/sup\u003e identified 32 \u003cem\u003eSr6\u003c/em\u003e-associated SNPs that intersected functional gene annotations in Chinese Spring. However, none of them overlapped with the candidate we identified in this study. We isolated \u003cem\u003eSr6\u003c/em\u003e using MutRenSeq.\u0026nbsp;After \u003cem\u003eYr5/YrSP\u003c/em\u003e and \u003cem\u003eYr7\u003c/em\u003e, \u003cem\u003eSr6\u003c/em\u003e is the fourth all-stage cloned wheat rust R gene containing a BED domain. However, such domain integrations are not uncommon in angiosperms. The rice resistance gene \u003cem\u003eXa1\u003c/em\u003e was identified as a BED-containing NLR\u003csup\u003e41\u003c/sup\u003e and recent genomic analyses identified many NLRs that incorporate non-canonical, integrated domains (IDs)\u003csup\u003e42\u0026ndash;44\u003c/sup\u003e. The BED domain does appear to be required for resistance as Marchal et al.\u003csup\u003e39\u003c/sup\u003e noted that a single, induced SNP in the BED domain of \u003cem\u003eYr7\u003c/em\u003e led to loss of resistance. Nevertheless, little is known about the functional role of BED domains in immune receptors despite a thorough genome-wide comparative evolutionary analysis of zinc finger BED transcription factor genes in land plants\u003csup\u003e45\u003c/sup\u003e. In Arabidopsis, the non-NB-LRR gene \u003cem\u003eDAYSLEEPER\u003c/em\u003e encodes a BED domain that was shown to bind DNA\u003csup\u003e46\u003c/sup\u003e. In the context of immunity, IDs are known to act as decoys for pathogen effectors. For example, an integrated WRKY domain in Arabidospsis R gene \u003cem\u003eRRS1-R\u003c/em\u003e binds bacterial effectors that target WRKY transcription factors\u003csup\u003e47\u003c/sup\u003e. It is unclear whether BED domains serve a similar purpose in ETI. However, the identification of a BED domain within leaf rust resistance gene \u003cem\u003eRph15\u003c/em\u003e from barley suggests that different rust pathogen species adapted to distinct hosts may have effectors targeting similar transcription factor domains\u003csup\u003e48\u003c/sup\u003e. The BED domain in \u003cem\u003eSr6\u003c/em\u003e appeared to be conserved across numerous accessions, not unlike the CC domain, so a potential role in signalling cannot be ruled out.\u003c/p\u003e \u003cp\u003eOur in-silico structural modelling and analysis did not show an association between the full-length structures or diverse N terminal structures of SR6, SR13, SR21 and temperature sensitivity of resistance. Whereas \u003cem\u003eSr13\u003c/em\u003e encodes a classic CC-NB-LRR, \u003cem\u003eSr21\u003c/em\u003e and the \u003cem\u003eSR9\u003c/em\u003e allelic series encode NLR proteins with a unique N terminus comprised of five helices bundle\u003csup\u003e49\u003c/sup\u003e, and \u003cem\u003eSr6\u003c/em\u003e falls into the zinc finger BED domain containing R protein encoding gene subset. Interestingly, we observed that all α3 helices from these proteins were the closest unit to their respective LRR domains. It is consistent with previous studies showing that the EDVID motif in the α3 helix from the CC domain of CNL protein SR35 interacts with its LRR domain to form a conserved EDVID-LRR\u003csup\u003eR\u0026minus;cluster\u003c/sup\u003e interface. It is considered as an evolutionarily conserved stabilization mechanism of CNL resistosomes and is important in mediating resistance-induced cell death\u003csup\u003e50\u003c/sup\u003e. Our observation implies both the zinc finger BED-containing group (SR6, YR5a, RPH15, XA1) and SR21 and the SR9 group could potentially share this conserved mechanism. Assuming this is the case, the possible correlation between the temperature sensitivity of the resistance and the distinct N terminal structure of the three proteins in the current study is low.\u003c/p\u003e \u003cp\u003eTransgenic complementation of susceptible Westonia plants with \u003cem\u003eSr6\u003c/em\u003e was successful in transferring resistance against \u003cem\u003ePgt\u003c/em\u003e. Reduced resistance at high temperatures was also demonstrated in transgenic plants despite maintained expression of \u003cem\u003eSr6\u003c/em\u003e, supporting a post-transcriptional or post-translational basis to temperature response. RNA analysis of the same transgenic plant transitioned between different temperatures may provide enhanced resolution of temperature dependent expression activity in future studies. The presence of the \u003cem\u003eSr6\u003c/em\u003e sequence in all transgenic progeny was unexpected and may be related to multiple insertions of the transgene, some non-functional. However, this is yet to be experimentally determined. The observation of residual resistance a in number of T\u003csub\u003e1\u003c/sub\u003e plants at high temperature may be related to transgene copy number or genomic insertion site, leading to over or under expression caused by position effects\u003csup\u003e51\u003c/sup\u003e. Further expression analysis on these resistance-retaining plants is needed to uncover the true mechanism.\u003c/p\u003e \u003cp\u003eTemperature has been shown to affect the relative abundance of transcript isoforms. Takabatake et al.\u003csup\u003e52\u003c/sup\u003e found that the tobacco (\u003cem\u003eNicotiana glutinosa\u003c/em\u003e) \u003cem\u003eN\u003c/em\u003e gene, which encodes an NLR, generates two alternative transcripts: a complete form encoding the full protein, and a truncated form encoding a protein lacking most of the LRR; both transcripts accumulated at 20\u0026deg;C, a permissive temperature, but not at 30\u0026deg;C, a non-permissive temperature in tobacco mosaic virus (TMV)-inoculated leaves. However, the levels of the complete isoform were always higher just before resistance-related cell death. Like \u003cem\u003eSr6\u003c/em\u003e, the \u003cem\u003eN\u003c/em\u003e gene can be reversibly inactivated, and this phenomenon is maintained in heterologous transgenic systems such as tomato and \u003cem\u003eN. benthamiana\u003c/em\u003e, indicating a conservation of signalling components\u003csup\u003e53\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNLRs are not the only class of R genes that exhibit temperature dependency. \u003cem\u003eYr36\u003c/em\u003e, conferring partial resistance to wheat stripe rust (caused by \u003cem\u003eP. striiformis\u003c/em\u003e f. sp. \u003cem\u003etritici\u003c/em\u003e), is more effective at higher greenhouse temperatures (25\u0026ndash;30⁰C) and encodes a kinase with a START lipid binding domain\u003csup\u003e54\u003c/sup\u003e. Fu et al.\u003csup\u003e54\u003c/sup\u003e found that \u003cem\u003eYr36\u003c/em\u003e generates multiple non-functional transcripts with truncations, and the functional isoform encoding the complete protein was upregulated at higher temperatures, mirroring the enhanced resistance seen at such temperatures. Stripe rust infection did not have a significant effect on functional transcript abundance compared to that of temperature. There is growing evidence that alternative splicing is one of the ways in which plants respond to temperature, as found in Arabidopsis with \u003cem\u003eLATE ELONGATED HYPOCOTYL\u003c/em\u003e (\u003cem\u003eLHY\u003c/em\u003e) which displayed temperature-associated isoform switching\u003csup\u003e55\u003c/sup\u003e. Similarly, the expression of \u003cem\u003eSr21\u003c/em\u003e was enhanced at higher temperatures, and was shown to encode 10 splice isoforms, albeit within the 3\u0026rsquo;UTR. However, three specific isoforms had increased abundance at 24⁰C compared to 16⁰C\u003csup\u003e24\u003c/sup\u003e. \u003cem\u003eSr13\u003c/em\u003e is also more effective at higher temperatures (25⁰C), but unlike \u003cem\u003eSr21\u003c/em\u003e, transcript abundance was not affected by temperature\u003csup\u003e23\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBased on the above, temperature-dependent resistance appears to be mediated primarily by transcriptional and post-transcriptional regulation. However, post-translational regulation has also been implicated; Zhu et al.\u003csup\u003e18\u003c/sup\u003e proposed temperature sensitivity was mediated by levels of inactive versus active protein conformations at varying temperatures, citing evidence of mutant R proteins that acquired heat stability, allowing them to remain in an active state at usually non-permissive temperatures. Additionally, little is known about the involvement of downstream signalling elements. In the case of \u003cem\u003eSr13\u003c/em\u003e, only PR gene expression was affected by temperature and not \u003cem\u003eSr13\u003c/em\u003e expression per se. Kiraly et al.\u003csup\u003e56\u003c/sup\u003e suggested that in the case of the tobacco N protein, the expression of downstream signalling components was also impaired at high temperatures. Clearly, the disease resistance pathway involves a myriad of regulatory and signalling components, some of which could be influenced to different degrees by temperature.\u003c/p\u003e \u003cp\u003eFor both \u003cem\u003eSr21\u003c/em\u003e and \u003cem\u003eSr13\u003c/em\u003e, upregulation of pathogenesis related (PR) genes was observed in resistant plants at higher temperatures only when inoculated with \u003cem\u003ePgt\u003c/em\u003e. PR genes were not upregulated in susceptible plants. This was further demonstrated by the results of this study, which showed similar upregulation of these genes at higher temperature. However, shifts in gene expression in response to temperature were less pronounced in \u003cem\u003eSr21-\u003c/em\u003ebearing lines. Overall, these results indicate the expression response of such PR genes is determined not by absolute temperature, but by activity of the \u003cem\u003eSr\u003c/em\u003e gene under permissive conditions.\u003c/p\u003e \u003cp\u003eWheat lines with temperature-sensitive \u003cem\u003eSr6\u003c/em\u003e, effective at lower temperatures, displayed largely unique differentially expressed genes and pathways compared to wheat lines with \u003cem\u003eSr13\u003c/em\u003e and \u003cem\u003eSr21\u003c/em\u003e, which are both more effective at higher temperatures. PR genes associated with the SA pathway were upregulated in wheat lines with all three genes relative to the controls under low temperatures for \u003cem\u003eSr6\u003c/em\u003e and high temperatures for \u003cem\u003eSr13\u003c/em\u003e and \u003cem\u003eSr21\u003c/em\u003e. The expression of these PR genes was much higher in plants with \u003cem\u003eSr6\u003c/em\u003e compared to the other two genes. This is consistent with the strong resistance phenotype of \u003cem\u003eSr6\u003c/em\u003e compared to the intermediate phenotype of \u003cem\u003eSr13\u003c/em\u003e and \u003cem\u003eSr21\u003c/em\u003e. Our study revealed unique pathways that are expressed in wheat lines with \u003cem\u003eSr6\u003c/em\u003e compared to \u003cem\u003eSr13\u003c/em\u003e and \u003cem\u003eSr21\u003c/em\u003e at different temperatures. Differential gene expression analysis revealed unique genes that are expressed during the onset of resistance or susceptibility. Several genes upregulated at low temperature in LMPG-\u003cem\u003eSr6\u003c/em\u003e were also upregulated either during susceptibility or at high temperature in \u003cem\u003eSr13\u003c/em\u003e and \u003cem\u003eSr21\u003c/em\u003e lines. To further explore the details of these host pathogen interactions and possible molecular changes, study of these genes at the protein level will be necessary. Similarly, genes associated with susceptibility require further study along with pathogen virulence factors. Our study highlights key factors to consider for temperature-dependent experiments on \u003cem\u003eSr\u003c/em\u003e gene expression: the effect of races, the frequency of alternative transcripts, overall expression, and regulation of PR genes and the currently neglected effect of pre-inoculation temperature.\u003c/p\u003e \u003cp\u003eThe cloning of \u003cem\u003eSr6\u003c/em\u003e adds to the suite of temperature-sensitive, cloned R genes in wheat. Further work will help to decipher the possibly shared mechanisms of temperature sensitivity underlying these genes and to what advantage, if any, temperature sensitivity has in plant immunity, keeping in mind that temperature effects will vary between controlled experiments and in the field. Additionally, the cloning of \u003cem\u003eSr6\u003c/em\u003e can expedite marker assisted screening and breeding programs in places where \u003cem\u003eSr6\u003c/em\u003e is still effective.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003ePlant materials for\u003c/b\u003e \u003cb\u003eSr6\u003c/b\u003e \u003cb\u003ecloning\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSeven M\u003csub\u003e3\u003c/sub\u003e putative point mutations for \u003cem\u003eSr6\u003c/em\u003e in Chinese Spring*5/Red Egyptian 2D (CS/RE 2D substitution line) were used in RenSeq analysis, namely, 3704-6, 3706-7, 3981-4, 4002-6, 4190-6, 5047-4, and 5140-5. F\u003csub\u003e3\u003c/sub\u003e lines derived from reciprocal crosses of CS \u0026times; CS/RE 2D were used in genetic analysis and confirmation of a diagnostic marker developed from the gene candidate. Fifteen seedlings per line were screened with \u003cem\u003ePgt\u003c/em\u003e race 21\u0026thinsp;\u0026minus;\u0026thinsp;2,3,7 (University of Sydney Culture no. 216) and resistant plants had IT 0;. The Australian cultivar Westonia was used for transformation, and T\u003csub\u003e1\u003c/sub\u003e progeny were assessed for validation of the candidate gene.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMutant generation\u003c/h2\u003e \u003cp\u003eSeeds of two batches (1,200 and 1,500) of chromosome substitution line CS/RE 2D were treated by 0.5 or 0.6% ethyl methanesulfonate (EMS), respectively, as described in Zhang \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e57\u003c/sup\u003e. Homozygous susceptible point mutants from seven different plants were used in subsequent R gene enrichment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMutRenSeq\u003c/h2\u003e \u003cp\u003eHigh molecular weight (HMW) DNA was prepared from leaves of non-infected seedlings of mutant and WT lines of CS/RE 2D using the phenol-chloroform method as described in Lagudah et al.\u003csup\u003e58\u003c/sup\u003e. DNA quality was assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and 0.8% agarose gels. Target R gene enrichment was performed using the \u003cem\u003eTriticeae\u003c/em\u003e RenSeq Bait Library V1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/steuernb/MutantHunter\u003c/span\u003e\u003cspan address=\"https://github.com/steuernb/MutantHunter\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) as described in Steuernagel et al.\u003csup\u003e59\u003c/sup\u003e. Subsequent steps of the pipeline were carried out as described in Hewitt et al.\u003csup\u003e60\u003c/sup\u003e using the MuTrigo package (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/TC-Hewitt/MuTrigo\u003c/span\u003e\u003cspan address=\"https://github.com/TC-Hewitt/MuTrigo\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for SNP calling and candidate search. Re-assembly using reads pooled from all samples (to increase coverage and enhance contiguity) was performed using the BioKanga package v4.4.2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/csiro-crop-informatics/biokanga\u003c/span\u003e\u003cspan address=\"https://github.com/csiro-crop-informatics/biokanga\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to trim raw reads with \u003cem\u003ebiokanga filter\u003c/em\u003e, assemble reads with \u003cem\u003ebiokanga assemb\u003c/em\u003e, and create scaffolds with \u003cem\u003ebiokanga scaffold\u003c/em\u003e using option \u003cem\u003e-P 1600\u003c/em\u003e, with all other parameters as default\u003csup\u003e61\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eConfirmation of SNPs and domain prediction\u003c/h2\u003e \u003cp\u003ePCR amplification of coding regions was performed on HMW DNA of mutants and WT. Products were cloned using a TOPO\u0026trade; XL-2 vector cloning kit (Invitrogen, Mulgrave, VIC, Australia). Plasmid DNA from at least four colonies per product was isolated using an ISOLATE II Plasmid Mini Kit (Bioline, Alexandria, NSW, Australia) and was then Sanger sequenced using primers listed in Supplementary Table\u0026nbsp;6. Coding sequences and translation were predicted using FGENESH (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.softberry.com/berry.phtml\u003c/span\u003e\u003cspan address=\"http://www.softberry.com/berry.phtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and amino acid substitutions were confirmed on alignment to mRNA sequence using CodonCode Aligner v8.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.codoncode.com/aligner/\u003c/span\u003e\u003cspan address=\"https://www.codoncode.com/aligner/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). NB-LRR motif annotations were created using NLR-Parser (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/steuernb/NLR-Parser\u003c/span\u003e\u003cspan address=\"https://github.com/steuernb/NLR-Parser\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and Zfn-BED domain prediction was performed using SMART (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl.de\u003c/span\u003e\u003cspan address=\"http://smart.embl.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with added PFAM (v8) domains\u003csup\u003e62\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSr6\u003c/b\u003e \u003cb\u003etransformation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA 8,160 bp genomic sequence of \u003cem\u003eSr6\u003c/em\u003e, including all introns, 2 kb upstream of 5\u0026prime;UTR and 1 kb downstream of the putative stop codon, encompassing the native promoter and terminator, was synthesized and cloned (Epoch Life Sciences, Missouri City, TX, USA) into \u003cem\u003eNot\u003c/em\u003eI/\u003cem\u003eSgs\u003c/em\u003eI-digested binary vector VecBarIII. Agrobacterium strain AGL1 was used to carry the construct and the \u003cem\u003eSr6\u003c/em\u003e gene was introduced into cv. Westonia using the Agrobacterium-transformation protocol\u003csup\u003e63\u003c/sup\u003e and phosphinothricin as the selective agent. Two independent transgenic plants (T\u003csub\u003e0\u003c/sub\u003e) carrying the \u003cem\u003eSr6\u003c/em\u003e transgene were recovered as confirmed with both PCR markers \u003cem\u003eSr6STS1\u003c/em\u003e and \u003cem\u003eSr6STS2\u003c/em\u003e (Supplementary Table\u0026nbsp;6). Seed from both T\u003csub\u003e0\u003c/sub\u003e plants were harvested and sets of 25 T\u003csub\u003e1\u003c/sub\u003e seedlings were grown at low (18℃) and high (25℃) temperatures in growth cabinets along with WT and positive and negative controls. After two weeks, the WT, controls and T\u003csub\u003e1\u003c/sub\u003e plants were inoculated with \u003cem\u003ePgt\u003c/em\u003e race 21\u0026thinsp;\u0026minus;\u0026thinsp;0 (University of Sydney Culture no. 330). Phenotyping and photography of leaf samples were performed at 14 days post-inoculation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePlanting, inoculation, and sample collection for RNAseq\u003c/h2\u003e \u003cp\u003eNear-isogenic wheat lines carrying \u003cem\u003eSr6\u003c/em\u003e, \u003cem\u003eSr13\u003c/em\u003e and \u003cem\u003eSr21\u003c/em\u003e, along with recurrent parent LMPG-6\u003csup\u003e64\u003c/sup\u003e were planted in coarse vermiculite in plastic pots (5 x 5 cm). Twenty seeds of a given line were planted in each pot. The North American wheat stem rust differential set\u003csup\u003e12\u003c/sup\u003e was planted for each treatment. Temperature treatments were conducted under high (25\u0026thinsp;\u0026minus;\u0026thinsp;22℃) and low (18\u0026thinsp;\u0026minus;\u0026thinsp;15℃) regimes (15-hr day/9-hr night) in controlled-environment cabinets. Pathogen treatments included spraying with either \u003cem\u003ePgt\u003c/em\u003e urediniospores mixed with Soltrol\u0026reg; or only Soltrol as a mock-inoculated treatment. Ten-day-old wheat seedlings were inoculated with urediniospores of \u003cem\u003ePgt\u003c/em\u003e isolate 59KS19 (race MCCFC, avirulent to \u003cem\u003eSr6\u003c/em\u003e, \u003cem\u003eSr13\u003c/em\u003e, and \u003cem\u003eSr21\u003c/em\u003e) according to previously described conditions\u003csup\u003e65\u003c/sup\u003e. A total of 20 plants of each line were included in each temperature and pathogen treatment. The entire experiment was repeated three times with different planting and inoculation dates. RNAseq was performed for each genotype at each temperature and inoculation treatment at two time points: 24 and 72 hours after inoculation. A total of six primary leaves were collected for RNAseq at each timepoint, temperature, and inoculation treatment. Altogether 96 samples were collected and subjected to RNAseq.\u003c/p\u003e \u003cp\u003ePrimary leaf tissue was collected at 24 h and 72 h post inoculation for RNAseq and qPCR experiments. Young leaves were collected in 2 ml flat bottom Eppendorf tubes. All samples were flash frozen in liquid nitrogen and stored at -80℃ for successive experiments. Seedling infection types on unharvested primary leaves were observed and recorded 14 days post-inoculation\u003csup\u003e66\u003c/sup\u003e. Infection types (IT) were rated on a 0\u0026ndash;4 scale with \u0026lsquo;0\u0026rsquo; indicating no visible symptoms and \u0026lsquo;4\u0026rsquo; being completely susceptible. IT greater than or equal to \u0026lsquo;3\u0026rsquo; were considered susceptible and less than \u0026lsquo;3\u0026rsquo; were considered resistant. IT were recorded on 5\u0026ndash;6 seedlings of each control and experimental line in each treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation, cDNA library construction, and sequencing\u003c/h2\u003e \u003cp\u003eThe collected tissues were subjected to RNA extraction using Qiagen plant RNeasy kits. On-column DNase digestion (30 min) was performed to remove possible DNA contamination. RNA was eluted from each sample using 50 \u0026micro;l of elution buffer and quality-checked on a Nanodrop ND-1000 spectrophotometer and agarose gel electrophoresis. Pre-screened RNA samples were subjected to TrueSeq and RiboZero library preparation at the University of Minnesota Genomics Center and quality checked on Bioanalyzer chips (Agilent Technologies). Prepared libraries were first sequenced on a NovaSeq 6000 S1 flow cell to obtain 100 bp PE reads with a target of 35\u0026nbsp;million reads per sample followed by a second sequencing run on a NovaSeq 6000 S4 flow cell to obtain 150 PE reads with a target of 37\u0026nbsp;million reads per sample.\u003c/p\u003e \u003cp\u003eThe sequence reads were quality checked using FastQC and quality trimmed to 100 bp PE by removing barcodes and Illumina adapter sequences. An index of high-confidence protein coding sequences obtained from the IWGSC RefSeq annotation v1.1 was created with kallisto 0.46.1\u003csup\u003e67\u003c/sup\u003e. The transcript sequences were also aligned to this index using kallisto (\u003cem\u003equant\u003c/em\u003e command) with 1,000 bootstraps of the PE reads to obtain alignment statistics. Alignment counts were further examined for differentially expressed genes (DEGs) in the R package edgeR\u003csup\u003e68\u003c/sup\u003e considering log fold change\u0026thinsp;\u0026ge;\u0026thinsp;2 and a false discovery rate (FDR) of \u0026lt;\u0026thinsp;0.05 by comparing infected lines to both mock-inoculated near-isogenic lines and inoculated-LMPG. An exon was retained when it was expressed\u0026thinsp;\u0026gt;\u0026thinsp;1 count per million (CPM) in at least three samples. Several DEGs were validated through qPCR. Genes unique to all three lines along with common genes with similar expression patterns in all three lines were selected for qPCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA analysis and qPCR\u003c/h2\u003e \u003cp\u003eFor qPCR, RNA isolated from leaf tissue at 0, 1 and 3 dpi were reverse transcribed to cDNA using protoScript\u0026reg; II Reverse Transcriptase (NEB). The reactions were carried out using probe-based IDT primers, 6FAM and IBFQ quencher, using 18S and GADPH as internal controls for gene expression analysis. TaqMan gene expression master mix (Applied Biosystems; Foster City, CA) and ABI 7300 (Applied Biosystems) were used for qPCR. The expression analysis was conducted using 2\u003csup\u003e\u0026minus;ΔΔC\u003c/sup\u003e\u003csub\u003eT\u003c/sub\u003e to determine the relative quantification\u003csup\u003e69\u003c/sup\u003e. The fold expression of \u003cem\u003eSr\u003c/em\u003e lines was compared with the susceptible LMPG-6 lines at low and high temperature. A total of four upregulated genes from RNAseq were selected to confirm their relative expression. Additionally, pathogenesis-related (PR) resistance genes associated with the salicylic acid and jasmonic acid pathways were selected and tested (Supplementary Tables\u0026nbsp;5.1, 5.2). Six PR and four JA genes were tested to identify how the corresponding pathways were associated with resistance and various temperatures. For each near-isogenic line, the expression of the relevant resistance gene (\u003cem\u003eSr6\u003c/em\u003e, \u003cem\u003eSr13\u003c/em\u003e, or \u003cem\u003eSr21\u003c/em\u003e) was tested at day 1 and day 3 at both low and high temperature treatments.\u003c/p\u003e \u003cp\u003eRT-qPCR was carried out independently on transgenic \u003cem\u003eSr6\u003c/em\u003e material using an alternative method. First, leaf tissues from each sample were frozen in liquid nitrogen or dry ice immediately after sampling; RNA was isolated using a RNeasy\u0026reg; Plant Mini Kit (QIAGEN, Chadstone Center, VIC, Australia) according to the manufacturer\u0026rsquo;s protocol, and used in first-strand DNA synthesis in 20 \u0026micro;L reactions using a Superscript\u0026reg; III reverse transcriptase kit (Life Technologies, Mulgrave, VIC, Australia). After the reverse transcript reaction, 3 \u0026micro;L of 10 ng/\u0026micro;L cDNA product was used for qPCR using a C1000 TouchTM thermocycler with the CFX96TM Real-Time System (Bio-Rad). qPCR conditions included an initial denaturation at 95\u0026deg;C for 3 min; 40 cycles of denaturation at 95\u0026deg;C for 10 s and annealing/elongation at 60\u0026deg;C for 30 s, followed by a melt step range of 65\u0026ndash;95\u0026deg;C with increments of 0.5\u0026deg;C. The wheat housekeeping gene TaCON was used as the reference gene for each qRT-PCR experiment\u003csup\u003e70\u003c/sup\u003e. qPCR primers specific for \u003cem\u003eSr6\u003c/em\u003e (forward: \u003cem\u003e5\u0026rsquo;-GTCAATAGCGCCGAGTGTAAG-3\u0026rsquo;\u003c/em\u003e, reverse: \u003cem\u003e5\u0026rsquo;-GGTCTGATGGCTGAATTACTGG-3\u0026rsquo;\u003c/em\u003e) were used to measure relative gene expression using three technical replicates for each sample. δCq mean values were calculated and standard errors were determined. Gene expression values were log (base 2)-transformed. Boxplots of aggregated relative expression values for each temperature regime and disease phenotype were generated using \u003cem\u003egeom_boxplot\u003c/em\u003e and \u003cem\u003egeom_point\u003c/em\u003e functions of R package ggplot2 (v3.3.6). Welch\u0026rsquo;s two-tailed unpaired t-tests were performed using the \u003cem\u003et.test\u003c/em\u003e function from base R.\u003c/p\u003e \u003cp\u003eRNA for rapid amplification of cDNA ends (RACE) was extracted from leaves of unchallenged, three-week-old seedlings of CS/RE 2D grown at ambient temperature (20\u0026ndash;25⁰C) using the RNeasy Plant Mini Kit (Qiagen); 5\u0026rsquo; and 3\u0026rsquo; RACE was conducted using a SMARTer RACE 5\u0026rsquo;/3\u0026rsquo; Kit (Clontech, Mountain View, CA, USA). RACE products were cloned using a TOPO\u0026trade; XL-2 vector cloning kit (Invitrogen). Plasmids were isolated from 10 colonies per product using an ISOLATE II Plasmid Mini Kit (Bioline) and Sanger sequenced.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGene annotation and pathway analysis\u003c/h2\u003e \u003cp\u003ePathway analysis and biological functions of differentially expressed genes were conducted via GeneOntology\u003csup\u003e71\u003c/sup\u003e, AgrigoV2\u003csup\u003e72\u003c/sup\u003e, and ShinyGo\u003csup\u003e73\u003c/sup\u003e. AgrigoV2 was used to identify the gene networks and pathways associated with the DEGs in different NILs at low or high temperature. GeneOntology software was used to categorize the DEGs into gene, family, protein, and species groups. The distribution of DEGs according to functional processes were represented in pie charts (Supplementary Figs.\u0026nbsp;10 and 11). ShinyGo was used to plot the DEGs across the chromosomes and calculate the statistical significance of various genomic regions\u003csup\u003e73\u003c/sup\u003e. Graphical enrichment analysis was completed using ShinyGo where query genes were mapped to the list of genes in particular pathways (Supplementary Figs.\u0026nbsp;12\u0026ndash;13, 17\u0026ndash;20). Fold enrichment was calculated by dividing the percentage of genes in the query list associated with a given pathway by the corresponding percentage of the genes in the background. FDR indicated the chances of fold enrichment and was calculated according to the Hypergeometric test\u003csup\u003e73\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePrimer design and reference comparison\u003c/h2\u003e \u003cp\u003eLocus-specific primers were designed manually based on polymorphisms in alignments between \u003cem\u003eSr6\u003c/em\u003e and high-scoring BLAST hits with Chinese Spring IWGSC RefSeq v1.0 (CSv1). DNA was isolated from leaves of F\u003csub\u003e3\u003c/sub\u003e lines using the method described in Ellis et al.\u003csup\u003e74\u003c/sup\u003e conducted on a Microlab NIMBUS\u0026trade; liquid handling robot (Hamilton, Reno, NV). STS marker screens and gene amplification were carried out using primers listed in Supplementary Table\u0026nbsp;6. Products were checked by electrophoresis on 1% agarose gels.\u003c/p\u003e \u003cp\u003eMarker \u003cem\u003eXwmc453\u003c/em\u003e proximal to \u003cem\u003eSr6\u003c/em\u003e\u003csup\u003e32\u003c/sup\u003e was positioned on CSv1 chromosome 2D using BLAST v2.3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/books/NBK131777/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/books/NBK131777/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with the probe sequence retrieved from GrainGenes (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wheat.pw.usda.gov/GG3/\u003c/span\u003e\u003cspan address=\"https://wheat.pw.usda.gov/GG3/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Homologous \u003cem\u003eSr6\u003c/em\u003e sequences were identified from CSv1 and various reference assemblies of the wheat 10\u0026thinsp;+\u0026thinsp;Genome Project (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.10wheatgenomes.com/\u003c/span\u003e\u003cspan address=\"http://www.10wheatgenomes.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), listed in Adamski et al.\u003csup\u003e75\u003c/sup\u003e, using BLAST v2.9. Full protein sequences of Yr5 and Yr7 provided in confidence were aligned with Sr6 using Clustal Omega (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/Tools/msa/clustalo/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/Tools/msa/clustalo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The transcript assembly of wheat cv. Avocet R used was the same as that described in Hewitt et al.\u003csup\u003e60\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eNLR dendrogram construction\u003c/h2\u003e \u003cp\u003eNLR protein sequences with an N-terminal coiled-coil domain (CNL class) were taken from the NCBI database. Accession numbers are listed in Supplementary Table\u0026nbsp;7; 125 sequences were aligned using MUSCLE and a phylogenetic tree was generated using the UPGMA method in MEGA X (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://github.com/steuernb/MutantHunter\" target=\"_blank\"\u003ewww.megasoftware.net\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.megasoftware.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Distances were computed using the Poisson correction method and are in units of the number of amino acid substitutions per site.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eProtein structural modelling and analysis\u003c/h2\u003e \u003cp\u003eFull length protein structure, ATP and zinc ion binding regions in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB were predicted by AlphaFold3 via AlphaFold Server at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://golgi.sandbox.google.com/\u003c/span\u003e\u003cspan address=\"https://golgi.sandbox.google.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003csup\u003e38\u003c/sup\u003e. Protein structures in Supplementary Figs. S14 and S15 were predicted by AlphaFold2 using ColabFold (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://colab.research.google.com/github/sokrypton/ColabFold/blob/main/beta/AlphaFold2_advanced.ipynb\u003c/span\u003e\u003cspan address=\"https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/beta/AlphaFold2_advanced.ipynb\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e76,77\u003c/sup\u003e. Structural and sequence analyses were carried out with PyMOL v2.5.5 and CLC Sequence Viewer v8. Weblogo diagram in Supplementary Fig.\u0026nbsp;16C was generated at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://weblogo.berkeley.edu/logo.cgi\u003c/span\u003e\u003cspan address=\"https://weblogo.berkeley.edu/logo.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eReporting summary\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets and plant materials generated and analyzed in the current study are available from the corresponding authors. The data that support the findings of this study are openly available in NCBI, the mRNA sequence of \u003cem\u003eSr6\u003c/em\u003e have been deposited at NCBI GenBank with accession number PP949235. Sequencing read data for MutRenSeq is deposited at DDBJ/EMBL/GenBank under BioProject PRJNA1127689. Other data are available within the paper and its Supplementary Information files. Source data are provided in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT.C.H., S.S., R.Mc., E.L., and P.Z. acknowledge the support from Grains Research and Development Corporation (GRDC), Australia. J.Z. acknowledges the support from the Australian Research Council (ARC) Early Career Industry Fellowship. M.N.R. acknowledges support from the USDA-ARS National Plant Disease Recovery System and a fellowship under the OECD Co-operative Research Programme: Biological Resource Management for Sustainable Agricultural Systems. T.C.H., J.Z., E.L., and P.Z. thank Dr. Chunhong Chen, CSIRO, for his excellent assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.Z., R.M., J.Z., S.S.: generation of mutants, disease phenotyping, and genotyping; T.H.: \u003cem\u003eSr6\u003c/em\u003e mutant data analysis and candidate validation. K.S.: qPCR, RNAseq analysis, and GO analysis of NILs; D.B.: generation of transgenic plants; P.Z, S.S.: growth, scoring and genotyping of transgenic plants; J.Z., Q.W.: Protein structure prediction and analysis; J.Y.: qPCR analysis of transgenic material; N.U.: created transcript assembly; P.B.: processed RNAseq data; K.S., M.R.: disease phenotyping of NILs; C.P.: provided Landmark sequence assembly before its publication; E.L., M.R., R.M., P.Z. designed and supervised the study; T.H., K.S., J.Z., M.R., R.M., E.L., P.Z. drafted the manuscript and all co-authors provided edits.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMcIntosh RA, Wellings CR, Park RF (1995) Wheat Rusts: An Atlas of Resistance Genes. CSIRO Publishing, Melbourne\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao L, Rouse MN, Mihalyov PD, Bulli P, Pumphrey MO, Anderson JA (2017) Genetic characterization of stem rust resistance in a global spring wheat germplasm collection. Crop Sci 57:2575\u0026ndash;2589\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeonard KJ, Szabo LJ (2005) Stem rust of small grains and grasses caused by \u003cem\u003ePuccinia graminis\u003c/em\u003e. Mol Plant Pathol 6:99\u0026ndash;111\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoko T, Bender CM, Prins R, Pretorius ZA (2018) Yield loss associated with different levels of stem rust resistance in bread wheat. Plant Dis 102:2531\u0026ndash;2538\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoelfs AP (1978) USDA Misc Publ No 1363. U.S. Department of Agriculture, Washington, DC\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRussell GE (1978) \u003cem\u003ePlant breeding for pest and disease resistance: Studies in the agricultural and food sciences\u003c/em\u003e. (Imprint: Butterworth-Heinemann, ISBN: 9781483192369\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRouse MN, Wanyera R, Njau P, Jin Y (2011) Sources of resistance to stem rust race Ug99 in spring wheat germplasm. Plant Dis 95:762\u0026ndash;766\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMourad AMI, Sallam A, Belamkar V, Wegulo S, Bowden R, Jin Y, Mahdy E, Bakheit B, El-Wafaa AA, Poland J et al (2018) Genome-wide association study for identification and validation of novel SNP markers for \u003cem\u003eSr6\u003c/em\u003e stem rust resistance gene in bread wheat. 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Nat Methods 19:679\u0026ndash;682\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"NLR, Puccinia graminis, temperature sensitivity, Triticum spp","lastPublishedDoi":"10.21203/rs.3.rs-4674841/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4674841/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe wheat stem rust pathogen, \u003cem\u003ePuccinia graminis\u003c/em\u003e f. sp. \u003cem\u003etritici\u003c/em\u003e (\u003cem\u003ePgt\u003c/em\u003e), has caused devastating crop losses worldwide. Several stem rust resistance (\u003cem\u003eSr\u003c/em\u003e) genes display temperature-dependent immune responses. \u003cem\u003eSr6\u003c/em\u003e-mediated resistance is enhanced at lower temperatures whereas \u003cem\u003eSr13\u003c/em\u003e and \u003cem\u003eSr21\u003c/em\u003e resistances are enhanced at higher temperatures. Here we report cloning of \u003cem\u003eSr6\u003c/em\u003e by mutagenesis and resistance gene enrichment and sequencing (MutRenSeq), showing it to encode an NLR protein with an integrated BED domain. \u003cem\u003eSr6\u003c/em\u003e temperature sensitivity was also transferred to wheat plants transformed with the \u003cem\u003eSr6\u003c/em\u003e transgene. Differential gene expression analysis using near-isogenic wheat lines inoculated with \u003cem\u003ePgt\u003c/em\u003e at varying temperatures revealed that genes upregulated in the low-temperature-effective \u003cem\u003eSr6\u003c/em\u003e response differed significantly from those upregulated in the high-temperature-effective responses associated with \u003cem\u003eSr13\u003c/em\u003e and \u003cem\u003eSr21\u003c/em\u003e. Understanding the molecular mechanisms and pathways involved in temperature sensitivity can inform future strategies for deployment and engineering of genetic resistance in response to a changing climate.\u003c/p\u003e","manuscriptTitle":"Divergent molecular pathways govern temperature-dependent wheat stem rust resistance genes Sr6, Sr13 and Sr21","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-16 08:05:05","doi":"10.21203/rs.3.rs-4674841/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"282a12e0-9fdb-4d76-bed0-9b01c31c0f21","owner":[],"postedDate":"July 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34537117,"name":"Biological sciences/Genetics/Plant genetics"},{"id":34537118,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-05-29T07:07:36+00:00","versionOfRecord":{"articleIdentity":"rs-4674841","link":"https://doi.org/10.1038/s41467-025-60030-x","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-05-28 04:00:00","publishedOnDateReadable":"May 28th, 2025"},"versionCreatedAt":"2024-07-16 08:05:05","video":"","vorDoi":"10.1038/s41467-025-60030-x","vorDoiUrl":"https://doi.org/10.1038/s41467-025-60030-x","workflowStages":[]},"version":"v1","identity":"rs-4674841","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4674841","identity":"rs-4674841","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}