Mutation of PUB21 in tomato leads to reduced susceptibility to necrotrophic fungi

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Visser, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6473041/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background Cultivated tomato is susceptible to necrotrophic pathogens Botrytis cinerea and Alternaria solani . No dominant resistance against these pathogens has been reported in wild relatives of tomato. Results Through screening of a tomato Micro-Tom EMS population we identified a mutant that showed decreased susceptibility to both necrotrophic fungi. Previously, we reported a mutation in the tomato PUB17 gene as the cause of reduced susceptibility in this mutant. Surprisingly, M4 progeny of one M3 plant homozygous for the pub17 mutation showed segregation with some plants displaying an even higher level of resistance than the pub17 mutant. This highly resistant progeny was shown to contain a mutation in tomato PUB21 in addition to the mutation in PUB17 . The role of PUB21 as a susceptibility factor for both necrotrophic fungi was confirmed in RNAi-silenced and CRISPR-mutated transformants. Conclusions In this study we identified a new PUB gene, SlPUB21 , involved in susceptibility of tomato to necrotrophic pathogens. We showed that mutation of this gene resulted in increased resistance against these pathogens. Tomato EMS PUB21 Susceptibility gene (S-gene) Botrytis cinerea Alternaria solani Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Botrytis cinerea is a necrotrophic fungal pathogen that poses a significant threat to a wide range of more than 1400 plant species, including tomato (Elad et al, 2016 ). This pathogen can cause severe damage to crops both pre- and post-harvest, leading to significant economic losses (Dean et al, 2012 ; Elad et al, 2004 ; Williamson et al, 2007 ). The success of B. cinerea as a pathogen can be attributed to its broad range of virulence factors, high reproductive capacity, and the lack of dominant resistance genes in its hosts (Bi et al, 2023 ; Leisen et al, 2021 ; Williamson et al, 2007 ). Thus, finding a way to mitigate the infection caused by B. cinerea is a pressing issue. Efforts have been made to understand the infection process of B. cinerea on plant hosts to identify key points of vulnerability. The fungus infects the host plant by secreting a multitude of cell death inducing proteins (CDIPs), suppressing the host autophagic response and manipulating the balance between autophagy and apoptosis (Bi et al, 2023 ; Leisen et al, 2021 ; Veloso & van Kan, 2018 ). This allows the fungus to grow and reach a critical biomass, before releasing compounds that trigger apoptotic cell death and enabling the spread of the infection. Since tomato as a crop is susceptible to infection by B. cinerea , tomato breeders have attempted to identify sources of resistance. Thus far, no gene providing dominant resistance has been identified in tomato. Hence, sources used by breeders for partial resistance against B. cinerea have relied on quantitative trait loci (QTLs) found in wild relatives of tomato such as Solanum chilense , S. peruvianum , S. chmielewskii , S. pimpinellifolium , S. habrochaites and S. neorickii (Chetelat & Stamova, 1999 ; Egashira et al, 2000 ; Nicot et al, 2002 ; ten Have et al, 2007 ). However, none of these QTLs individually or combined could provide the same level of resistance as in the wild relatives (Davis et al, 2009 ; Finkers et al, 2007 ). An alternative approach to developing resistance against B. cinerea , and potentially other necrotrophic pathogens such as Alternaria solani , is focused on disabling susceptibility (S) genes. An S-gene refers to any gene that plays a role in facilitating infection and maintaining the pathogen-host compatibility. Van Schie & Takken ( 2014 ) classified S-genes into three categories based on the distinct stage of the plant-pathogen interaction they are involved in: host recognition and entry, immune system suppression, and compatibility maintenance to support pathogen proliferation. Since plant S-genes provide advantages to the pathogen, deactivating or reducing the expression of these genes should result in increased plant resistance. Indeed, accumulating evidence demonstrates that a loss-of-function mutation of S-genes in the host may result in resistance against different pathogens (Jiang & Yu, 2016 ; Santillán Martínez et al, 2020 ; Sun et al, 2017 ), making S-genes compelling targets in the search for resistance against necrotrophic pathogens such as B. cinerea . In Arabidopsis more than 60 genes involved in the progression of B. cinerea infection have been identified (a.o. Castillo et al, 2019 ; Costa et al, 2015 ; Cui et al, 2019 ; Jiang & Yu, 2016 ; Jurkowski et al, 2004 ; Nishimura et al, 2003 ; van Schie & Takken, 2014 ; Wang et al, 2015 ). However, many S-genes have crucial functions in plant physiological processes, and therefore, a loss-of-function mutation could potentially result in a decrease of fitness of the plant (Hanika et al, 2021 ). To identify novel S-genes that facilitate the infection by necrotrophic fungi, we adopted a forward genetics approach by screening a tomato EMS population for resistance against the necrotrophic pathogen B. cinerea . Previously, we described an EMS mutant (named M2042) that carries a knock-out mutation in the U-box E3 ubiquitin ligase PUB17 gene (Ramírez Gaona et al, 2023 ). The loss of function of the PUB17 gene led to reduced susceptibility towards B. cinerea as well as the necrotrophic fungus A. solani . However, some of the tomato mutant plants manifested slight autonecrosis depending on genetic background. However, crossing of the PUB17 mutant with different genetic backgrounds successfully abolished the spontaneous autonecrosis in some of the progeny (Ramírez Gaona et al, 2023 ), thus increasing the potential of its use in breeding. In this study we report the identification of an additional mutated S-gene in one M3 progeny of the M2042 family that conferred resistance to B. cinerea and A. solani . The responsible gene mutation was identified and verification that the gene functions as an S-gene was obtained through the analysis of RNAi and CRISPR transformants. Results An EMS-induced mutation in PUB21 (Solyc11g006030) is correlated with reduced susceptibility to Botrytis A Micro-Tom EMS population (Yan et al., 2021 ) was previously screened for reduced susceptibility to B. cinerea . Mutant M2042 displayed intermediate resistance (IR) towards the fungus, and it was shown that the responsible mutation had occurred in gene Solyc02g072080 encoding PUB17 (Ramírez Gaona et al, 2023 ). Remarkably, when testing progeny of M3 plant M2042-1-3, which was homozygous for the pub17 mutation (pedigree in Fig. S1), we observed segregation of extreme resistant (R) plants among the M4 progeny. The extreme resistant plants not only displayed smaller lesions but also developed small light green leaves (Fig. 1 a) (a.o. M2042-1-3-10, M2042-1-3-11 and M2042-1-3-14) when compared to the IR plants (M2042-1-3-5).This suggested the involvement of another mutation in addition to the one in PUB17 . To identify the additional mutation, segregating F2 populations were produced from a cross between MM and M4 plant M2042-1-3-10 (pedigree in Fig. S1b). Five F1 plants were selfed to produce F2 seeds. A total of 205 F2 plants were screened using a B. cinerea detached leaf assay (DLA) to distinguish between three different classes: extreme resistant (small lesion = R), intermediate resistant (medium lesion = IR) and susceptible (large lesion = S) plants. Subsequently, plants showing either very small or very large lesion diameters were selected and reinoculated to confirm resistance or susceptibility. The three groups (S, IR, R) showed a ratio of 9:6:1 suggesting the involvement of two distinct, genetically unlinked loci. Next, two extreme pools were constructed for a bulked segregant analysis (BSA): extreme resistant (M2042-3R) and susceptible (M2042-3S), with 13 and 14 plants per pool, respectively. DNA of the selected plants was mixed per pool and subjected to Whole Genome Sequencing (WGS). A total of 2,028,009 SNPs were obtained (compared to the reference genome of tomato cv. Heinz) and filtered to identify causal candidate genes. Bioinformatic analyses were performed to select sequence variants absent from wild-type Micro-Tom plants, present in high frequency in M2042-3R and in low frequency in M2042-3S pool (Fig. 1 b). In pool M2042-3R, the stop-gain mutation on chromosome 2 previously identified in PUB17 was present (Ramírez Gaona et al, 2023 ; Fig. 1 c), along with an additional mutation on the top of chromosome 11 (Fig. 1 b). A mutation consisting of a T→A transversion was observed in gene Solyc11g006030 (Fig. 1 c) at position 890 of the coding region, resulting in a premature stop codon L297* (Fig. 1 d). Individual plants per pool were genotyped to confirm the correlation between the mutation and Botrytis resistance. The Solyc11g006030 gene product belongs to the Plant U-box (PUB) protein family and has similarity with Arabidopsis At5g37490 (PUB21/CMPG5) and potato StPUB21 (Fig. S2). It is therefore proposed as the tomato homolog of At PUB21 . To determine the relative expression levels of both genes PUB17 and PUB21 , a RT-qPCR was performed using wild-type (WT) MT plants and M4 progeny showing strong resistance (double mutant M2042-1-3-14). Samples of mock- and B. cinerea- inoculated leaves were collected at three time points, 0, 24 and 48 hours post inoculation (hpi). The expression of both PUB17 and PUB21 was highly induced upon infection with B. cinerea in WT MT (Fig. 2 ). The large variation observed in PUB21 expression in MT at 24 and 48 hpi can be attributed to the lower absolute expression level of PUB21 reported for WT Heinz leaves under non-inoculated conditions (0.15 RPKM) as compared to PUB17 gene expression (19.71 RPKM), as shown in the Tomato Expression Database (TED; Fei et al, 2006 ). Contrasting with WT MT, in the double pub17 / pub21 mutant M2042-1-3-14 both PUB17 and PUB21 expression remained uninduced after inoculation with B. cinerea (Fig. 2 ). Botrytis resistance in single pub17 , single pub21 and double pub17/pub21 mutants To determine the individual effect of the mutation in PUB21 , F2 plants from the cross between MM and EMS double mutant M2042-1-3-10 were genotyped for MT mutations d (dwarf) and sp (self-pruning) (Marti et al, 2006) with a PCR using primers shown in Table S1. Selected individuals were self-fertilized to produce progeny with homozygous MM alleles for D and Sp , in order to obtain progeny with a plant morphology more similar to MM than the dwarf tomato MT. Furthermore, they were selected for being homozygous double pub17 / pub21 mutants or homozygous single pub21 mutants with homozygous MM allele of PUB17 (Fig. S1b). Subsequently, three F3 and three F4 single pub21 lines along with two F4 double mutant lines were screened for Botrytis resistance and compared with four F4 and one F5 single pub17 mutant lines (Fig. S1a) and control MM plants using the DLA approach. Average lesion diameters per line were calculated (Fig. 3 ). Overall, the single pub17 and pub21 mutants showed comparable average lesion diameters, differing significantly from control MM plants. The single mutants both displayed intermediate Botrytis resistance. The double pub17 / pub21 mutants showed stronger reduction in average lesion diameters, significantly different from the single mutants, indicating strong Botrytis resistance. Silencing of PUB21 by RNAi results in increased resistance against B. cinerea To confirm that the PUB21 gene is indeed an S-gene towards B. cinerea , knock-down and knock-out transformants were produced using RNAi and CRISPR editing, respectively. Two RNAi constructs targeting PUB21 were made: RNAi fragment 1 (195 bp) targeting the region between the U-box domain and ARM repeats, and RNAi fragment 10 (205 bp) targeting the ARM repeats domain (Fig. 4 ). A total of 18 RNAi transformants was obtained with construct RNAi1 and 17 with construct RNAi10. After transfer of transformants to the greenhouse it was noticed that some of the RNAi transformants showed slight autonecrosis on the leaves. T2 progeny was obtained from these primary transformants and individual plants were selected for T3 seed production based on the presence of NPTII from the silencing construct. Relative expression levels of PUB21 were determined in the silenced lines using qRT-PCR (Table 1 , Fig. 5 a). As mentioned above, absolute expression level of PUB21 is low in wild-type plants, which complicated accurate measurement of silencing levels. Despite this, several RNAi transformant T3 families showing a lower level of expression than control plants were identified. Table 1 RNAi PUB21 transformants and codes of T2 and T3 progeny. RNAi T1 transformant* T2 plant T3 Family Code Relative PUB21 expression level RNAi 1–5 TV191052-4 TV202215 TV15 0.89 RNAi 1–7 TV191053-5 TV202218 TV18 0.31 RNAi 1–21 TV191059-20 TV202231 TV31 0.58 RNAi 10 − 9 TV191072-16 TV202234 TV34 0.29 RNAi 10–26 TV191077-11 TV202241 TV41 0.45 RNAi 10–26 TV191077-7 TV202240 TV40 (no NPTII) ND Moneymaker control WT 1.0 * RNAi 1 transformants were obtained with a silencing construct containing RNAi fragment 1 (Fig. 4 ), while RNAi 10 transformants were obtained with RNAi fragment 10. ND, not determined. The T3 families were inoculated with B. cinerea in a DLA. The two controls, MM and TV202240 (no NPTII present), showed similar B. cinerea lesion diameters (Fig. 5 b). In contrast, significantly smaller B. cinerea lesions were observed on leaves from T3 families TV202218, TV202234 and TV202241. To generate CRISPR/Cas9 mutations in PUB21 , a transformation construct was designed with 3 sgRNAs targeting different regions of the gene (Fig. 4 ). MM was transformed with this construct and 37 transformants were obtained. The transformants were genotyped by sequencing PCR products obtained using a primer pair flanking the first and last sgRNAs, leading to the identification of 2 transformants with mutant alleles at the target site of sgRNA1. Only small indels were observed in plants 30 (allele 1) and 35 (allele 2) (Fig. 4 ). A summary of the identified mutations is provided in Table 2 . The CRISPR plants homozygous for mutant PUB21 allele 1 or 2 had a height similar to MM, with slightly smaller leaves. Small spontaneous necrotic spots appeared in older leaves of these plants. Table 2 Progeny of PUB21 CRISPR mutants. WT, wild type; NA, not applicable. T1 plant # T2 family-plant # T3 family Code Mutant alleles Mutation 30 TV191084-16 TV202269 TV69 Homozygous mutant allele 1 4-bp deletion 30 TV191085-32 TV202278 TV78 Homozygous WT allele NA 35 TV191086-6 TV202281 TV81 Heterozygous mutant allele 1 4-bp deletion 35 TV191086-30 TV202285 TV85 Heterozygous mutant allele 2 1-bp insertion (T) 35 TV191086-9 TV202283 TV83 Homozygous WT allele NA Homozygous mutant T3 progeny was obtained from T1 transformants 30 and 35 (Table 2 ). The T3 families were subjected to a B. cinerea DLA. MM plants and PUB21 CRISPR T3 families TV202278 (TV78) and TV202283 (TV83) containing only wild-type PUB21 alleles, were used as the susceptible controls. Compared with the negative controls (MM and T3 families TV78 and TV83), all T3 plants homozygous for CRISPR-induced mutations in PUB21 exhibited significantly smaller lesion diameters upon B. cinerea inoculation in the DLA (Fig. 6 ), demonstrating that knocking out PUB21 in MM background leads to increased resistance to B. cinerea . The homozygous family TV69 displayed lesions ~ 27% smaller than the WT MM lesions at 3 dpi. Meanwhile the T3 families TV81 and TV85, segregating for the 4-bp deletion and 1-bp deletion respectively, were split into three groups: homozygous mutants (TV81M, TV85M), heterozygous mutants (TV81H, TV85H) and homozygous wild-type (TV81WT, TV85WT). The homozygous TV81M plants exhibited lesions ~ 32% smaller than the WT MM lesions at 3 dpi, while lesions from TV85M plants were ~ 26% smaller than the WT lesions. Consequence of mutations on PUB21 protein structure/architecture To compare the sequence features of the PUB21 proteins in the stable CRISPR mutant with the original PUB21 EMS mutant, a multiple sequence alignment was made of the predicted proteins (Fig. S3). The alignment revealed that both CRISPR mutant alleles had frameshift mutations and early stop codons halfway through the U-box domain, leading also to a complete loss of the ARM domain (amino acids 192–364) (Fig. 7 ). Meanwhile the EMS mutant contained an intact U-box domain (amino acids 22–94) with an early stop codon within the ARM domain, leading to a truncated third ARM repeat and a loss of the fourth ARM repeat. Susceptibility of PUB21 mutants to other pathogens To investigate whether mutating the PUB21 gene influences susceptibility to other tomato pathogens, disease assays were performed on the pub21 mutant with the necrotrophic fungus A. solani . The original single pub17 M2042 mutant (Ramírez Gaona et al, 2023 ) as well as the EMS-derived pub17 / pub21 double mutant showed significantly reduced lesion diameters (Fig. 8 ), with the pub17 / pub21 double mutant showing smaller lesions than the original M2042 mutant. In addition, pub21 CRISPR T3 mutant families and pub21 T3 RNAi lines were subjected to a DLA with A. solani . Homozygous mutant T3 plants of CRISPR families TV202285 (TV85M), TV202269 (TV69) and TV202281 (TV81M) clearly developed significantly smaller lesions than the negative controls (Fig. 9 ). The RNAi T3 families TV202218, TV202234 and TV202241 displayed significantly smaller lesions compared to the negative controls, MM and T3 family TV202240, while these did not show any significant difference among each other (Fig. S4). When tested with the obligate biotrophic tomato powdery mildew pathogen Pseudoidium neolycopersici , plants of both the original pub17 mutant M2042 and pub21 CRISPR T3 family TV202269 were as susceptible as the control plants of MT or MM (Fig. S5). Discussion Tomato genes PUB17 and PUB21 are susceptibility genes towards necrotrophic fungi In this study we report that tomato PUB21 , a U-box E3 ubiquitin ligase (Azevedo et al, 2001 ), acts as a S-gene during infection with the necrotrophic fungi B. cinerea and A. solani . In Arabidopsis, 64 PUB genes have been described (Andersen et al, 2004 ) and categorized into 7 classes based on the presence/absence of domains other than the highly conserved U-box domain, such as Armadillo repeats (ARM) (Trenner et al, 2022 ). PUB21 belongs to Class IV with the presence of ARM repeats located at the C-terminus. M4 progeny of EMS mutant family M2042-1-3, which showed stronger resistance against B. cinerea than the original mutant, contained a mutated PUB21 allele in addition to the mutation in PUB17 . The reduced susceptibility observed in the individual F3 and F4 pub21 mutants was comparable to that observed in the F4 and F5 pub17 . The role of PUB21 in conferring susceptibility to B. cinerea , independent of PUB17 , was confirmed through CRISPR mutation and RNAi silencing of the wild-type allele in MM background. Meanwhile, the pub17/pub21 double mutants showed even lower susceptibility to B. cinerea. These results demonstrate that mutations in PUB17 and PUB21 have an additive effect on Botrytis resistance. In addition, similar to the pub17 mutants, tomato pub21 mutants also showed lower susceptibility to the necrotroph A. solani . The pub21 mutation in the EMS mutant resulted in the loss of the two C-terminal Armadillo repeats while the CRISPR-induced mutations resulted in a truncated U-box domain along with the complete loss of the ARM domain. The U-box is a highly conserved domain known mainly for its involvement in pairing with specific E2 proteins providing a certain level of target specificity (Trenner et al, 2022 ). Mutations in the U-box domain have been reported to cause a loss of E3 activity (Turek et al, 2018 ). On the other hand, the ARM repeats are involved in protein-protein interactions for plant development, morphogenesis and hormone signaling (Coates, 2007 ). Specifically, ARM repeats present in PUB proteins provide a secondary level of specificity when it comes to interacting with E2 ubiquitin-conjugating enzymes, as E2-E3 pairing is determined by both the U-box and the ARM domain (Samuel et al, 2006 ; Turek et al, 2018 ). Therefore, loss of these domains would hinder the ubiquitination process as specific binding of proteins targeted by PUB21 is lost. While the CRISPR mutants have a truncated U-box domain and a complete loss of the ARM repeats, two essential domains for proper E3 ligase activity, the reduced susceptibility is similar to that of the EMS mutant. This suggests that the loss of the ARM repeats is enough to lead to lower susceptibility. PUB21 and its close homolog PUB20 act as regulators of immunity Studies on the function of PUB21 in tomato or PUB21 orthologs in other plant species are scarce. Comparison of homologs may provide valuable information on the evolutionary relationship between species and insights on conserved biological pathways. In Arabidopsis, PUB21 is also known as CMPG5. The closest homolog to PUB21 in Arabidopsis is PUB20/CMPG1 (Fig. S2). Yee and Goring ( 2009 ) performed a microarray study in Arabidopsis measuring transcript level changes after pathogen-derived elicitor treatment. AtPUB21 was induced by treatment with various pathogens, including B. cinerea , Phytophthora infestans , Pseudomonas syringae pv. phaseolicola , as well as several PAMP (pathogen-associated molecular pattern) signals (Yee & Goring, 2009 ). Given the response to pathogens and pathogen-derived molecules, AtPUB21 was suggested to be a regulator of immunity. The different time points of induction in response to different pathogen inoculations suggests that this regulatory function of AtPUB21 depends on the stage of infection. A recent article by Yi et al ( 2024 ) showed that both AtPUB20 and AtPUB21 act as negative regulators of immunity at the early stage after Pseudomonas syringae pv. tomato DC3000 invasion. Both pub20 and pub21 mutants showed increased resistance to this bacterial pathogen. In soybean ( Glycine max ) a PUB20 gene ( Glyma.14G212200 ) and a PUB21 -like gene ( Glyma.18G042100 ; Fig. S2) were among the highest upregulated genes after inoculation with the necrotrophic fungus Sclerotinia sclerotiorum (Wei et al, 2022 ). On the other hand, Nicotiana benthamiana CMPG1 and the N. tabacum ortholog ACRE74 have been reported as a positive regulators of immunity against Cladosporium fulvum based on the observation that RNAi-silenced plants exhibited reduced cell death induction triggered after recognition of C. fulvum effector Avr9 by resistance gene Cf-9 (González-Lamothe et al, 2006 ). The phylogenetic tree (Fig. S2) shows that the PUB20 and PUB21 proteins from Solanaceous species form two separate clusters, while AtPUB20 and AtPUB21 are in their own subgroup, suggesting that homology between the tomato and Arabidopsis PUB21 genes is not enough to infer a function from one species to the other. In the case of A. thaliana , the amino acid sequences of AtPUB20 and AtPUB21 display 56% identity and 72% similarity. Importantly, they do not interact with the same targets, as AtPUB20 interacts with AGB1 (GTP-binding protein beta 1), while AtPUB21 does not (Kobayashi et al, 2012 ). AGB1 is reported to be required for resistance against the necrotrophic fungus Plectosphaerella cucumerina (Delgado-Cerezo et al, 2012 ). Evolutionary pressures may have prompted PUB gene E3 ligase diversification to manage different biotic or abiotic stresses. Role of tomato PUB21 in programmed cell death As the loss of function of tomato PUB21 leads to lower susceptibility to necrotrophic fungi, we can deduce that SlPUB21 functions as a negative regulator of immunity for this class of pathogens. The presence of spontaneous necrotic spots in older leaves of the pub21 mutants suggest a regulatory function in programmed cell death (PCD). PCD plays a pivotal role in enabling a successful infection of necrotrophic pathogens. Two types of PCD can be distinguished: apoptosis and autophagy. While the activation of either pathway leads to the appearance of necrotic plant tissue, the final outcome of each pathway leads to different effects on plant health. Research on the necrotrophic fungi Sclerotinia sclerotiorum and B. cinerea has shown that infection benefited from apoptotic PCD triggered upon inoculation with the pathogen (Dickman et al, 2001 ). Veloso & van Kan ( 2018 ) proposed that necrotrophic pathogens have the ability to temporarily suppress autophagic cell death in the early phases of infection in order to proliferate and infect the host plant. It remains to be determined which host target genes are involved in PCD regulation (Bi et al, 2023 ; Leisen et al, 2021 ). We infer that PUB21 is one of those host genes. In the pub21 mutant the balance is shifted towards autophagic PCD. PUB21 may either positively regulate apoptosis or negatively regulate autophagy. We have shown that loss of ARM repeats of PUB21 is sufficient to reduce susceptibility to necrotrophs. Although the specific targets of PUB21 are unknown, studying the protein-protein interactions might reveal the target protein(s) of PUB21 and thereby explain how it contributes to PCD. PUB17 and PUB21 are involved in different PCD pathways An increased resistance against both Botrytis and Alternaria was observed in the pub17 / pub21 EMS double mutant compared to the single pub17 or pub21 mutants. Therefore, the double mutant shows an additive effect, suggesting that each gene targets a different part of the complex cell death process. If both genes would be involved in the same pathway we would expect similar lesion diameters when comparing the double mutant with the single mutants. However, the loss of two genes involved in different aspects of the regulation of PCD, leading to stronger resistance against the necrotrophic pathogens, is accompanied by stronger pleiotropic effects on development/growth of the plant. Further testing of the double mutant and the single mutants is thus required to elucidate the roles of both PUB17 and PUB21 in PCD. Conclusion Our results show that SlPUB21 is a key player in the susceptibility of tomato plants to the necrotrophic pathogens B. cinerea and A. solani . The mutation of SlPUB21 provides broad-spectrum resistance to these pathogens without displaying severe pleiotropic effects. The double mutant of pub17/pub21 in tomato displayed a higher level of resistance, however, it also exhibited reduced plant size. We surmise that SlPUB21 is involved in programmed cell death regulation in a different way than SlPUB17. Materials and methods Plant material Two different tomato cultivars were used for this study: cv Micro-Tom (MT) and cv. Moneymaker (MM). MT seeds were obtained from Beekenkamp Plants B.V. (Maasdijk, The Netherlands). MT was chosen to generate an EMS population (Yan et al, 2021 ) because of its small size, short life cycle and the possibility to grow at high density (Meissner et al, 1997 ). Full details on the development of the MT-EMS population have been described by Ramírez Gaona et al ( 2023 ). Disease assays Detached leaf assays (DLA) for both B. cinerea strain B05.10 and A. solani isolate CBS 143772 were performed following the methods described by Ramírez Gaona et al ( 2023 ). The powdery mildew disease assay with Pseudoidium neolycopersici isolate On-Ne was performed by spraying whole plants with a conidiospore suspension of 2.5 x 10 4 spores per ml, as described by Bai et al ( 2003 ). Identification of mutant gene by Bulked Segregant Analysis combined with Whole Genome Sequencing (BSA-WGS) EMS mutant M2042 (Ramírez Gaona et al, 2023 ) showing reduced susceptibility to B. cinerea was selfed until M4 plants were obtained (Fig. S1). Individual M4 plants were selfed for M5 seed production (M2042-1-1-17 and M2042-1-2-12) and/or crossed with MM to develop F1 seeds (M2042-1-2-12, M2042-1-3-10). F1 plants were selfed and F2 seeds were collected. F2 plants were tested for susceptibility to B. cinerea by DLA to build two pools, consisting of resistant and susceptible F2 plants. Mutants obtained from the F2 generation were selfed to establish F3 lines, and subsequently F4 and F5 lines (Fig. S1a). DNA of the resistant and susceptible F2 plants was isolated following the method described by Ramírez Gaona et al ( 2023 ) and subsequently pooled equimolarly, resulting in two DNA pools M2042-3R and M2042-3S. Two additional pools had been made previously from the F2 progeny of M2042-1-2-12, M2042S (susceptible pool) and M2042R (intermediate resistant pool) (Ramírez Gaona et al, 2023 ); together with a pool of wild-type Micro-Tom plants (MTWT) these were used as controls for the PUB17 mutation. Whole genome sequencing of the DNA pools was done as outlined in Ramírez Gaona et al ( 2023 ). SNP detection was performed using SAMtools on the reads that were mapped to the tomato Heinz reference genome (version SL2.50). For each SNP the number of reads containing the reference allele (Heinz) and the number of reads with the alternative allele for each pool were recorded. Subsequent calculations and SNP filtering were performed as described by Ramírez Gaona et al. ( 2023 ). Each chromosome was inspected for the presence of large difference of percentage of alternative allele per SNP position between M2042-3R and M2042-3S (∆SNP = %ALT[R] - %ALT[S] > 50). On the short arm of chromosome 11 (SL2.50ch11:841716..840424), a large ∆SNP was observed between pools M2042-3R and M2042-3S. The next filtering consisted of selection of SNP positions in exons of annotated genes for which the alternative allele was present in the pools M2042-3R and M2042-3S of the extreme resistant M2042-1-3-10 but not in the MTWT pool, nor in the previously obtained resistant and susceptible pools (M2042R and M2042S) used to identify PUB17 (Ramírez Gaona et al, 2023 ). Gene expression level by RT-qPCR Gene expression levels were determined by performing a RT-qPCR on plant cDNA synthesized using an iScript cDNA Synthesis kit (BioRad) on RNA extracted through an RNeasy Plant Mini Kit (Qiagen). Specific primers were developed for each gene (Table S1). Elongation factor 1 alpha ( Ef1α ) was used as reference gene. RT-qPCR was performed using a CFX96 Real-Time PCR machine (BioRad) with two technical replicates used per sample. Relative expression of PUB17 or PUB21 was calculated with the ΔΔC T method (Livak & Schmittgen, 2001 ). RNAi and CRISPR transformation for confirmation of candidate gene PUB21 RNAi constructs were generated using the binary vector pHellsgate8 (Helliwell and Waterhouse 2003); full details are described in Ramírez Gaona et al ( 2023 ). This vector contains a CaMV 35S promoter driving the expression of the inverted repeat and a kanamycin resistance gene as a selectable marker. Primers were designed for PUB21 to amplify fragments from tomato MM gDNA. Primer sequences are shown in Table S1. RNAi fragment 1 targets the region between the U-box domain and the Armadillo (ARM) repeats of the PUB21 protein, while the RNAi fragment 10 targets the ARM repeats domain. A CRISPR/Cas9 construct was developed to create deletions within the PUB21 coding sequence, using 3 sgRNAs alongside the Cas9 endonuclease gene and the NPTII plant selectable marker. The sgRNAs were designed as described by Santillán Martínez et al ( 2020 ). To select the best 3 sgRNAs, in addition to sgRNA scorer ( https://sgrnascorer.cancer.gov [Chari et al, 2017 ]), two additional tools were used: GPP sgRNA Designer ( https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design [Sanson et al, 2018 ]), and WU-CRISPR ( http://crispr.wustl.edu [Wong et al, 2015 ]). The construct was assembled using a Golden Gate cloning system (Engler et al, 2008 ) with plasmids from Addgene as described by Santillán Martínez et al ( 2020 ). The plasmids were cloned using E.coli DH5α. The two RNAi constructs and one CRISPR/Cas9 construct for PUB21 were transformed into electrocompetent Agrobacterium tumefaciens AGL1 + virG cells. Transformation of tomato cv. MM was carried out as previously described (Huibers et al, 2013 ). DNA was isolated from young leaves using CTAB buffer (1 M Tris-HCl pH 7.5, 0.5 M EDTA pH 8.0, 5 M NaCl, 2% CTAB) essentially as described by Doyle (1991). To confirm the integration of the T-DNAs of the silencing constructs in the genome of the RNAi transformants, a PCR was performed to detect the presence of the NPTII gene and 35S promoter using DreamTaq DNA polymerase (Thermo Scientific, Bleiswijk, The Netherlands). Primer sequences are shown in Table S1. To determine the presence of mutations in the CRISPR transformants PCRs were performed using primers flanking the sgRNA target sites. The PCR products were sent for sequencing to Macrogen Europe (Amsterdam, The Netherlands). Statistical analysis Data points for each DLA experiment were subjected to an ANOVA F-test using R studio v 1.1.463 (Allaire, 2012 ). The ANOVA test was followed by a Post Hoc test using Tukey HSD method to perform multiple pairwise-comparisons. Differences were considered significant at P < 0.05. Protein alignment and prediction tools Sequencing of the PUB21 EMS mutant and CRISPR T3 mutants was performed using primers covering the complete genomic sequence (Table S1). MM and MT DNA samples were taken along as controls. The PUB21 sequences were assembled by aligning the DNA fragments using the plasmid editor ApE (Davis & Jorgensen, 2022 ). Each complete DNA sequence was translated to RNA using the ExPASy translate tool ( https://web.expasy.org/translate/ [Gasteiger et al, 2003 ]). Protein alignment was done using the multiple sequences alignment program Clustal Omega provided by EMBL-EBI (Madeira et al, 2022 ). Protein domains were predicted using the ScanProsite tool (De Castro et al, 2006 ). The Arabidopsis PUB protein sequences were obtained from TAIR ( https://www.arabidopsis.org/ [Rhee et al, 2003 ]), while the rest of the PUB protein sequences were obtained from NCBI ( https://www.ncbi.nlm.nih.gov/ ). After protein alignment, MAFFT7 ( https://mafft.cbrc.jp/alignment/server/ ) was used to build the phylogenetic tree. Declarations Acknowledgements We thank Fien Meijer-Dekens and Bertus van der Laan for taking care of the plants in the greenhouse, and Jaap Wolters for providing inoculum of Alternaria solani . Author contributions YB, AMAW, JALvK and RGFV conceived the study. AvT performed the screening of the EMS population. MRG, DS and AvT performed the experiments. JALvK provided disease testing resources and protocols. MRG, AMAW and YB analysed the results. MRG wrote the manuscript with input from all co-authors. Funding This research was financially supported by grants from Foundation Topconsortium voor Kennis en Innovatie (TKI) Horticulture & Starting Materials projects EZ-2012-07 and TU-18015. Data availability The data generated and analyzed supporting the findings of the current work are available within the manuscript and its supplementary information files. Ethics approval and consent to participate Not applicable. Clinical trial number Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Allaire J. RStudio: integrated development environment for R. Boston, MA, 2012;770:165-171. Andersen P, Kragelund BB, Olsen AN, Larsen FH, Chua NH, Poulsen FM, Skriver K. Structure and biochemical function of a prototypical Arabidopsis U-box domain. J. Biol. Chem. 2004;279:40053-40061. Azevedo C, Santos-Rosa MJ, Shirasu K. The U-box protein family in plants. Trends Plant Sci. 2001 ; 6:354-358. Bai Y, Huang C-C, van der Hulst R, Meijer-Dekens F, Bonnema G, Lindhout P. QTLs for tomato powdery mildew resistance ( Oidium lycopersici ) in Lycopersicon parviflorum G1.1601 co-localize with two qualitative powdery mildew resistance genes. Mol. Plant-Microbe Interact. 2003;16:169-176. Bi K, Liang Y, Mengiste T, Sharon A. Killing softly: A roadmap of Botrytis cinerea pathogenicity. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6473041","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":450192016,"identity":"73f5b6cd-3e68-4749-b538-9316bea88245","order_by":0,"name":"Miguel Ramírez Gaona","email":"","orcid":"","institution":"Wageningen University \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"Miguel","middleName":"Ramírez","lastName":"Gaona","suffix":""},{"id":450192017,"identity":"cbbba2bf-dafc-48b6-9ec3-b996a3bf1791","order_by":1,"name":"Ageeth van Tuinen","email":"","orcid":"","institution":"Wageningen University \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"Ageeth","middleName":"van","lastName":"Tuinen","suffix":""},{"id":450192018,"identity":"5f9219b7-42f8-4263-9419-803ef1ad8827","order_by":2,"name":"Danny Schipper","email":"","orcid":"","institution":"Wageningen University \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"Danny","middleName":"","lastName":"Schipper","suffix":""},{"id":450192019,"identity":"f31661fe-213b-4800-a163-c9f10360e5ba","order_by":3,"name":"Richard G.F. 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(\u003cstrong\u003ea\u003c/strong\u003e) Three classes of response observed at 4 days after \u003cem\u003eB. cinerea\u003c/em\u003e inoculation (4 dpi) in wild-type MicroTom and M2042-derived M4 plants.\u003cem\u003e \u003c/em\u003e(\u003cstrong\u003eb\u003c/strong\u003e) SNP frequencies of the alternative alleles (compared to reference genome Heinz) in the extreme resistant and the susceptible F2 bulks of mutant M2042-1-3-10 on chromosome 11. (\u003cstrong\u003ec\u003c/strong\u003e) Chromosome 2 and Chromosome 11 SNP positions per bulk. REF, reference allele from Heinz; ALT, alternative allele; MTWT, wild type Micro-Tom bulk; M2042R, intermediate resistant bulk from M2042-1-2-12; M2042S, susceptible bulk from M2042-1-2-12 (Ramírez Gaona et al, 2023); M2042_3R, extreme resistant bulk from M2042-1-3-10; M2042_3S, susceptible bulk from M2042-1-3-10. (\u003cstrong\u003ed\u003c/strong\u003e) Tomato \u003cem\u003ePUB21 \u003c/em\u003e(Solyc11g006030) has 2 domains: U-box domain (amino acids 22-94) and ARM (armadillo) repeats (amino acids 192-364). The EMS mutation in the ARM repeats domain, indicated with an orange line, causes a premature stop codon L297*.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6473041/v1/9206649b9d5b153af1bf5bbf.png"},{"id":81822006,"identity":"da201da6-6a47-44b7-a256-83b54c5095c8","added_by":"auto","created_at":"2025-05-02 11:31:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":197408,"visible":true,"origin":"","legend":"\u003cp\u003eRelative gene expression level of \u003cem\u003ePUB17\u003c/em\u003e (\u003cstrong\u003ea\u003c/strong\u003e) and \u003cem\u003ePUB21\u003c/em\u003e (\u003cstrong\u003eb\u003c/strong\u003e) in wild-type Micro-Tom (MT) and \u003cem\u003eBotrytis\u003c/em\u003e-resistant double mutant plant M2042-1-3-14 (3-14) upon infection with \u003cem\u003eB. cinerea\u003c/em\u003e. hpi, hours post inoculation.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6473041/v1/a8d9e337cd35b23374100d81.png"},{"id":81822007,"identity":"9f2b87cc-54a7-4e53-9eaa-8ffc16211db6","added_by":"auto","created_at":"2025-05-02 11:31:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":368097,"visible":true,"origin":"","legend":"\u003cp\u003eAverage lesion diameters of single \u003cem\u003epub17\u003c/em\u003e and \u003cem\u003epub21\u003c/em\u003emutants and double \u003cem\u003epub17\u003c/em\u003e/\u003cem\u003epub21\u003c/em\u003e mutant F4 and F5 families compared with Moneymaker (MM) after \u003cem\u003eBotrytis\u003c/em\u003e infection. Results from 4 days post inoculation (dpi). Pedigree information of the mutant lines is shown in Fig S1.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6473041/v1/feec8679b0972d757541ab69.png"},{"id":81822011,"identity":"799b68d1-2e6e-44f9-9a28-dbf6fe8e3b39","added_by":"auto","created_at":"2025-05-02 11:31:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":178723,"visible":true,"origin":"","legend":"\u003cp\u003eRNAi fragments and guide RNA target sites in gene \u003cem\u003eSlPUB21\u003c/em\u003e(Solyc11g006030). The single exon of the wild-type \u003cem\u003ePUB21\u003c/em\u003e allele in cultivar Heinz is shown as a green arrow, with underneath the positions of two RNAi fragments for silencing, and three guide RNAs for CRISPR editing as blue arrows. Primers used for analysis of the EMS-induced mutation are shown as small yellow arrows, while the primers used to check for CRISPR-induced mutations are shown as small orange arrows. Sizes of deletions in the CRISPR transformants at the sgRNA1 target site are indicated as small blue boxes above the exon.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6473041/v1/dccee573af184405c1cacc91.png"},{"id":81822693,"identity":"7a78d36f-999e-409b-9ed0-79a33046420b","added_by":"auto","created_at":"2025-05-02 11:39:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":248994,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Relative expression levels of tomato \u003cem\u003ePUB21\u003c/em\u003ein RNAi T3 families (TV15-TV41) compared to untransformed Moneymaker (MM) as determined by qRT-PCR using \u003cem\u003eEF1α\u003c/em\u003e as reference gene. (\u003cstrong\u003eb\u003c/strong\u003e) Boxplot of \u003cem\u003eBotrytis cinerea\u003c/em\u003e lesion diameters on leaves from \u003cem\u003ePUB21 \u003c/em\u003eRNAi T3 families with the two controls (MM and TV202240) on the left, results from 4 dpi. Codes of the T3 families are explained in Table 1.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6473041/v1/42a8a334974dd81d63b9a672.png"},{"id":81822009,"identity":"aa3bbcf2-6435-400d-a8cd-26c001167101","added_by":"auto","created_at":"2025-05-02 11:31:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":220687,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplot of \u003cem\u003eBotrytis cinerea\u003c/em\u003elesion diameter sizes on leaves from \u003cem\u003ePUB21\u003c/em\u003eCRISPR mutant T3 plants of TV85M (homozygous for mutant allele 2), TV69 and TV81M (both homozygous for mutant allele 1), compared with negative controls Moneymaker (MM), TV85H (heterozygous for mutant allele 2), TV81H (heterozygous for mutant allele 1), non-mutant T3 families TV83 and TV78 and T3 plants from TV85WT, 4 days after inoculation. Different letters above the boxplots indicate significant differences, as calculated by Tukey HSD method (P\u0026lt; 0.05). H, heterozygous; WT, homozygous wild-type; M, homozygous mutant.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6473041/v1/932651652eda413237a1fffe.png"},{"id":81822008,"identity":"5f0424a5-265b-424c-b1d0-d2b212413a7f","added_by":"auto","created_at":"2025-05-02 11:31:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":94635,"visible":true,"origin":"","legend":"\u003cp\u003eProtein domains in mutant \u003cem\u003ePUB21\u003c/em\u003e alleles. WT, wild type tomato PUB21 protein; M2042, EMS pub21 mutant protein; Allele 1-2, CRISPR pub21 mutant proteins. Output obtained from the Scan Prosite tool (accessed on 06/06/23). Red vertical lines indicate early stop codons; red dashed horizontal lines indicate different amino acids than in the WT allele.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6473041/v1/814b5c17f2dd87eea3071dfc.png"},{"id":81823024,"identity":"64d7acd6-ba6d-4069-b151-306d7cb2cf9e","added_by":"auto","created_at":"2025-05-02 11:47:21","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":171417,"visible":true,"origin":"","legend":"\u003cp\u003eAverage lesion diameter sizes on leaves 5 days after inoculation (5 dpi) with \u003cem\u003eAlternaria solani\u003c/em\u003e. MM, Moneymaker; MT, Micro-Tom. Pedigree of the mutant plants shown in Fig S1.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6473041/v1/207ae084394b89bc0e17b30a.png"},{"id":81822020,"identity":"da719394-35b5-4aea-bd81-74cfac3fca2d","added_by":"auto","created_at":"2025-05-02 11:31:21","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":188678,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplot of \u003cem\u003eAlternaria solani\u003c/em\u003e lesion diameters on leaves from \u003cem\u003epub21\u003c/em\u003eCRISPR T3 families including controls. Abbreviations of T3 families explained in Table 2. Results from 7 days post inoculation (dpi). Different letters above the boxplots indicate significant differences, as calculated by Tukey HSD method (P\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6473041/v1/399336ff79f79c90f4cb32c8.png"},{"id":81823987,"identity":"a63b6720-fada-4d3f-ba1a-266cb9355ce3","added_by":"auto","created_at":"2025-05-02 12:03:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3324049,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6473041/v1/d6d0b808-3426-4480-a572-d3f83e238046.pdf"},{"id":81822692,"identity":"0f913644-c17f-4d43-beb6-d5ab1b539097","added_by":"auto","created_at":"2025-05-02 11:39:21","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2050668,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6473041/v1/7367a191648c56e5ca3a36a0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mutation of PUB21 in tomato leads to reduced susceptibility to necrotrophic fungi","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eBotrytis cinerea\u003c/em\u003e is a necrotrophic fungal pathogen that poses a significant threat to a wide range of more than 1400 plant species, including tomato (Elad et al, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This pathogen can cause severe damage to crops both pre- and post-harvest, leading to significant economic losses (Dean et al, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Elad et al, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Williamson et al, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The success of \u003cem\u003eB. cinerea\u003c/em\u003e as a pathogen can be attributed to its broad range of virulence factors, high reproductive capacity, and the lack of dominant resistance genes in its hosts (Bi et al, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Leisen et al, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Williamson et al, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Thus, finding a way to mitigate the infection caused by \u003cem\u003eB. cinerea\u003c/em\u003e is a pressing issue.\u003c/p\u003e \u003cp\u003eEfforts have been made to understand the infection process of \u003cem\u003eB. cinerea\u003c/em\u003e on plant hosts to identify key points of vulnerability. The fungus infects the host plant by secreting a multitude of cell death inducing proteins (CDIPs), suppressing the host autophagic response and manipulating the balance between autophagy and apoptosis (Bi et al, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Leisen et al, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Veloso \u0026amp; van Kan, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This allows the fungus to grow and reach a critical biomass, before releasing compounds that trigger apoptotic cell death and enabling the spread of the infection.\u003c/p\u003e \u003cp\u003eSince tomato as a crop is susceptible to infection by \u003cem\u003eB. cinerea\u003c/em\u003e, tomato breeders have attempted to identify sources of resistance. Thus far, no gene providing dominant resistance has been identified in tomato. Hence, sources used by breeders for partial resistance against \u003cem\u003eB. cinerea\u003c/em\u003e have relied on quantitative trait loci (QTLs) found in wild relatives of tomato such as \u003cem\u003eSolanum chilense\u003c/em\u003e, \u003cem\u003eS. peruvianum\u003c/em\u003e, \u003cem\u003eS. chmielewskii\u003c/em\u003e, \u003cem\u003eS. pimpinellifolium\u003c/em\u003e, \u003cem\u003eS. habrochaites\u003c/em\u003e and \u003cem\u003eS. neorickii\u003c/em\u003e (Chetelat \u0026amp; Stamova, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Egashira et al, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Nicot et al, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; ten Have et al, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). However, none of these QTLs individually or combined could provide the same level of resistance as in the wild relatives (Davis et al, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Finkers et al, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAn alternative approach to developing resistance against \u003cem\u003eB. cinerea\u003c/em\u003e, and potentially other necrotrophic pathogens such as \u003cem\u003eAlternaria solani\u003c/em\u003e, is focused on disabling susceptibility (S) genes. An S-gene refers to any gene that plays a role in facilitating infection and maintaining the pathogen-host compatibility. Van Schie \u0026amp; Takken (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) classified S-genes into three categories based on the distinct stage of the plant-pathogen interaction they are involved in: host recognition and entry, immune system suppression, and compatibility maintenance to support pathogen proliferation. Since plant S-genes provide advantages to the pathogen, deactivating or reducing the expression of these genes should result in increased plant resistance. Indeed, accumulating evidence demonstrates that a loss-of-function mutation of S-genes in the host may result in resistance against different pathogens (Jiang \u0026amp; Yu, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Santill\u0026aacute;n Mart\u0026iacute;nez et al, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sun et al, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), making S-genes compelling targets in the search for resistance against necrotrophic pathogens such as \u003cem\u003eB. cinerea\u003c/em\u003e. In Arabidopsis more than 60 genes involved in the progression of \u003cem\u003eB. cinerea\u003c/em\u003e infection have been identified (a.o. Castillo et al, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Costa et al, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Cui et al, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Jiang \u0026amp; Yu, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Jurkowski et al, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Nishimura et al, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; van Schie \u0026amp; Takken, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wang et al, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, many S-genes have crucial functions in plant physiological processes, and therefore, a loss-of-function mutation could potentially result in a decrease of fitness of the plant (Hanika et al, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo identify novel S-genes that facilitate the infection by necrotrophic fungi, we adopted a forward genetics approach by screening a tomato EMS population for resistance against the necrotrophic pathogen \u003cem\u003eB. cinerea\u003c/em\u003e. Previously, we described an EMS mutant (named M2042) that carries a knock-out mutation in the U-box E3 ubiquitin ligase \u003cem\u003ePUB17\u003c/em\u003e gene (Ram\u0026iacute;rez Gaona et al, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The loss of function of the \u003cem\u003ePUB17\u003c/em\u003e gene led to reduced susceptibility towards \u003cem\u003eB. cinerea\u003c/em\u003e as well as the necrotrophic fungus \u003cem\u003eA. solani\u003c/em\u003e. However, some of the tomato mutant plants manifested slight autonecrosis depending on genetic background. However, crossing of the \u003cem\u003ePUB17\u003c/em\u003e mutant with different genetic backgrounds successfully abolished the spontaneous autonecrosis in some of the progeny (Ram\u0026iacute;rez Gaona et al, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), thus increasing the potential of its use in breeding.\u003c/p\u003e \u003cp\u003eIn this study we report the identification of an additional mutated S-gene in one M3 progeny of the M2042 family that conferred resistance to \u003cem\u003eB. cinerea\u003c/em\u003e and \u003cem\u003eA. solani\u003c/em\u003e. The responsible gene mutation was identified and verification that the gene functions as an S-gene was obtained through the analysis of RNAi and CRISPR transformants.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eAn EMS-induced mutation in\u003c/b\u003e \u003cb\u003ePUB21\u003c/b\u003e \u003cb\u003e(Solyc11g006030) is correlated with reduced susceptibility to\u003c/b\u003e \u003cb\u003eBotrytis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA Micro-Tom EMS population (Yan et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) was previously screened for reduced susceptibility to \u003cem\u003eB. cinerea\u003c/em\u003e. Mutant M2042 displayed intermediate resistance (IR) towards the fungus, and it was shown that the responsible mutation had occurred in gene Solyc02g072080 encoding \u003cem\u003ePUB17\u003c/em\u003e (Ram\u0026iacute;rez Gaona et al, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Remarkably, when testing progeny of M3 plant M2042-1-3, which was homozygous for the \u003cem\u003epub17\u003c/em\u003e mutation (pedigree in Fig. S1), we observed segregation of extreme resistant (R) plants among the M4 progeny. The extreme resistant plants not only displayed smaller lesions but also developed small light green leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) (a.o. M2042-1-3-10, M2042-1-3-11 and M2042-1-3-14) when compared to the IR plants (M2042-1-3-5).This suggested the involvement of another mutation in addition to the one in \u003cem\u003ePUB17\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTo identify the additional mutation, segregating F2 populations were produced from a cross between MM and M4 plant M2042-1-3-10 (pedigree in Fig. S1b). Five F1 plants were selfed to produce F2 seeds. A total of 205 F2 plants were screened using a \u003cem\u003eB. cinerea\u003c/em\u003e detached leaf assay (DLA) to distinguish between three different classes: extreme resistant (small lesion\u0026thinsp;=\u0026thinsp;R), intermediate resistant (medium lesion\u0026thinsp;=\u0026thinsp;IR) and susceptible (large lesion\u0026thinsp;=\u0026thinsp;S) plants. Subsequently, plants showing either very small or very large lesion diameters were selected and reinoculated to confirm resistance or susceptibility. The three groups (S, IR, R) showed a ratio of 9:6:1 suggesting the involvement of two distinct, genetically unlinked loci. Next, two extreme pools were constructed for a bulked segregant analysis (BSA): extreme resistant (M2042-3R) and susceptible (M2042-3S), with 13 and 14 plants per pool, respectively. DNA of the selected plants was mixed per pool and subjected to Whole Genome Sequencing (WGS). A total of 2,028,009 SNPs were obtained (compared to the reference genome of tomato cv. Heinz) and filtered to identify causal candidate genes. Bioinformatic analyses were performed to select sequence variants absent from wild-type Micro-Tom plants, present in high frequency in M2042-3R and in low frequency in M2042-3S pool (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In pool M2042-3R, the stop-gain mutation on chromosome 2 previously identified in \u003cem\u003ePUB17\u003c/em\u003e was present (Ram\u0026iacute;rez Gaona et al, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), along with an additional mutation on the top of chromosome 11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). A mutation consisting of a T\u0026rarr;A transversion was observed in gene Solyc11g006030 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) at position 890 of the coding region, resulting in a premature stop codon L297* (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Individual plants per pool were genotyped to confirm the correlation between the mutation and \u003cem\u003eBotrytis\u003c/em\u003e resistance. The Solyc11g006030 gene product belongs to the Plant U-box (PUB) protein family and has similarity with Arabidopsis At5g37490 (PUB21/CMPG5) and potato StPUB21 (Fig. S2). It is therefore proposed as the tomato homolog of At\u003cem\u003ePUB21\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the relative expression levels of both genes \u003cem\u003ePUB17\u003c/em\u003e and \u003cem\u003ePUB21\u003c/em\u003e, a RT-qPCR was performed using wild-type (WT) MT plants and M4 progeny showing strong resistance (double mutant M2042-1-3-14). Samples of mock- and \u003cem\u003eB. cinerea-\u003c/em\u003einoculated leaves were collected at three time points, 0, 24 and 48 hours post inoculation (hpi). The expression of both \u003cem\u003ePUB17\u003c/em\u003e and \u003cem\u003ePUB21\u003c/em\u003e was highly induced upon infection with \u003cem\u003eB. cinerea\u003c/em\u003e in WT MT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The large variation observed in \u003cem\u003ePUB21\u003c/em\u003e expression in MT at 24 and 48 hpi can be attributed to the lower absolute expression level of \u003cem\u003ePUB21\u003c/em\u003e reported for WT Heinz leaves under non-inoculated conditions (0.15 RPKM) as compared to \u003cem\u003ePUB17\u003c/em\u003e gene expression (19.71 RPKM), as shown in the Tomato Expression Database (TED; Fei et al, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Contrasting with WT MT, in the double \u003cem\u003epub17\u003c/em\u003e/\u003cem\u003epub21\u003c/em\u003e mutant M2042-1-3-14 both \u003cem\u003ePUB17\u003c/em\u003e and \u003cem\u003ePUB21\u003c/em\u003e expression remained uninduced after inoculation with \u003cem\u003eB. cinerea\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBotrytis\u003c/b\u003e \u003cb\u003eresistance in single\u003c/b\u003e \u003cb\u003epub17\u003c/b\u003e, \u003cb\u003esingle\u003c/b\u003e \u003cb\u003epub21\u003c/b\u003e \u003cb\u003eand double\u003c/b\u003e \u003cb\u003epub17/pub21\u003c/b\u003e \u003cb\u003emutants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine the individual effect of the mutation in \u003cem\u003ePUB21\u003c/em\u003e, F2 plants from the cross between MM and EMS double mutant M2042-1-3-10 were genotyped for MT mutations \u003cem\u003ed\u003c/em\u003e (dwarf) and \u003cem\u003esp\u003c/em\u003e (self-pruning) (Marti et al, 2006) with a PCR using primers shown in Table S1. Selected individuals were self-fertilized to produce progeny with homozygous MM alleles for \u003cem\u003eD\u003c/em\u003e and \u003cem\u003eSp\u003c/em\u003e, in order to obtain progeny with a plant morphology more similar to MM than the dwarf tomato MT. Furthermore, they were selected for being homozygous double \u003cem\u003epub17\u003c/em\u003e/\u003cem\u003epub21\u003c/em\u003e mutants or homozygous single \u003cem\u003epub21\u003c/em\u003e mutants with homozygous MM allele of \u003cem\u003ePUB17\u003c/em\u003e (Fig. S1b). Subsequently, three F3 and three F4 single \u003cem\u003epub21\u003c/em\u003e lines along with two F4 double mutant lines were screened for \u003cem\u003eBotrytis\u003c/em\u003e resistance and compared with four F4 and one F5 single \u003cem\u003epub17\u003c/em\u003e mutant lines (Fig. S1a) and control MM plants using the DLA approach. Average lesion diameters per line were calculated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOverall, the single \u003cem\u003epub17\u003c/em\u003e and \u003cem\u003epub21\u003c/em\u003e mutants showed comparable average lesion diameters, differing significantly from control MM plants. The single mutants both displayed intermediate \u003cem\u003eBotrytis\u003c/em\u003e resistance. The double \u003cem\u003epub17\u003c/em\u003e/\u003cem\u003epub21\u003c/em\u003e mutants showed stronger reduction in average lesion diameters, significantly different from the single mutants, indicating strong \u003cem\u003eBotrytis\u003c/em\u003e resistance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSilencing of\u003c/b\u003e \u003cb\u003ePUB21\u003c/b\u003e \u003cb\u003eby RNAi results in increased resistance against\u003c/b\u003e \u003cb\u003eB. cinerea\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo confirm that the \u003cem\u003ePUB21\u003c/em\u003e gene is indeed an S-gene towards \u003cem\u003eB. cinerea\u003c/em\u003e, knock-down and knock-out transformants were produced using RNAi and CRISPR editing, respectively. Two RNAi constructs targeting \u003cem\u003ePUB21\u003c/em\u003e were made: RNAi fragment 1 (195 bp) targeting the region between the U-box domain and ARM repeats, and RNAi fragment 10 (205 bp) targeting the ARM repeats domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA total of 18 RNAi transformants was obtained with construct RNAi1 and 17 with construct RNAi10. After transfer of transformants to the greenhouse it was noticed that some of the RNAi transformants showed slight autonecrosis on the leaves. T2 progeny was obtained from these primary transformants and individual plants were selected for T3 seed production based on the presence of NPTII from the silencing construct. Relative expression levels of \u003cem\u003ePUB21\u003c/em\u003e were determined in the silenced lines using qRT-PCR (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). As mentioned above, absolute expression level of \u003cem\u003ePUB21\u003c/em\u003e is low in wild-type plants, which complicated accurate measurement of silencing levels. Despite this, several RNAi transformant T3 families showing a lower level of expression than control plants were identified.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRNAi \u003cem\u003ePUB21\u003c/em\u003e transformants and codes of T2 and T3 progeny.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRNAi T1 transformant*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT2 plant\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT3 Family\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCode\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRelative \u003cem\u003ePUB21\u003c/em\u003e expression level\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRNAi 1\u0026ndash;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTV191052-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTV202215\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTV15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRNAi 1\u0026ndash;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTV191053-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTV202218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTV18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRNAi 1\u0026ndash;21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTV191059-20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTV202231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTV31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRNAi 10\u0026thinsp;\u0026minus;\u0026thinsp;9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTV191072-16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTV202234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTV34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRNAi 10\u0026ndash;26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTV191077-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTV202241\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTV41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRNAi 10\u0026ndash;26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTV191077-7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTV202240\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTV40 (no NPTII)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eMoneymaker control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e* RNAi 1 transformants were obtained with a silencing construct containing RNAi fragment 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), while RNAi 10 transformants were obtained with RNAi fragment 10. ND, not determined.\u003c/p\u003e \u003cp\u003eThe T3 families were inoculated with \u003cem\u003eB. cinerea\u003c/em\u003e in a DLA. The two controls, MM and TV202240 (no NPTII present), showed similar \u003cem\u003eB. cinerea\u003c/em\u003e lesion diameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). In contrast, significantly smaller \u003cem\u003eB. cinerea\u003c/em\u003e lesions were observed on leaves from T3 families TV202218, TV202234 and TV202241.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo generate CRISPR/Cas9 mutations in \u003cem\u003ePUB21\u003c/em\u003e, a transformation construct was designed with 3 sgRNAs targeting different regions of the gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). MM was transformed with this construct and 37 transformants were obtained. The transformants were genotyped by sequencing PCR products obtained using a primer pair flanking the first and last sgRNAs, leading to the identification of 2 transformants with mutant alleles at the target site of sgRNA1. Only small indels were observed in plants 30 (allele 1) and 35 (allele 2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A summary of the identified mutations is provided in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The CRISPR plants homozygous for mutant \u003cem\u003ePUB21\u003c/em\u003e allele 1 or 2 had a height similar to MM, with slightly smaller leaves. Small spontaneous necrotic spots appeared in older leaves of these plants.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProgeny of \u003cem\u003ePUB21\u003c/em\u003e CRISPR mutants. WT, wild type; NA, not applicable.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT1 plant #\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT2 family-plant #\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT3 family\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCode\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMutant alleles\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMutation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTV191084-16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTV202269\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTV69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHomozygous mutant allele 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4-bp deletion\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTV191085-32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTV202278\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTV78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHomozygous WT allele\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTV191086-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTV202281\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTV81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHeterozygous mutant allele 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4-bp deletion\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTV191086-30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTV202285\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTV85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHeterozygous mutant allele 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1-bp insertion (T)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTV191086-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTV202283\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTV83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHomozygous WT allele\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eHomozygous mutant T3 progeny was obtained from T1 transformants 30 and 35 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The T3 families were subjected to a \u003cem\u003eB. cinerea\u003c/em\u003e DLA. MM plants and \u003cem\u003ePUB21\u003c/em\u003e CRISPR T3 families TV202278 (TV78) and TV202283 (TV83) containing only wild-type \u003cem\u003ePUB21\u003c/em\u003e alleles, were used as the susceptible controls.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCompared with the negative controls (MM and T3 families TV78 and TV83), all T3 plants homozygous for CRISPR-induced mutations in \u003cem\u003ePUB21\u003c/em\u003e exhibited significantly smaller lesion diameters upon \u003cem\u003eB. cinerea\u003c/em\u003e inoculation in the DLA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), demonstrating that knocking out \u003cem\u003ePUB21\u003c/em\u003e in MM background leads to increased resistance to \u003cem\u003eB. cinerea\u003c/em\u003e. The homozygous family TV69 displayed lesions\u0026thinsp;~\u0026thinsp;27% smaller than the WT MM lesions at 3 dpi. Meanwhile the T3 families TV81 and TV85, segregating for the 4-bp deletion and 1-bp deletion respectively, were split into three groups: homozygous mutants (TV81M, TV85M), heterozygous mutants (TV81H, TV85H) and homozygous wild-type (TV81WT, TV85WT). The homozygous TV81M plants exhibited lesions\u0026thinsp;~\u0026thinsp;32% smaller than the WT MM lesions at 3 dpi, while lesions from TV85M plants were ~\u0026thinsp;26% smaller than the WT lesions.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eConsequence of mutations on PUB21 protein structure/architecture\u003c/h2\u003e \u003cp\u003eTo compare the sequence features of the PUB21 proteins in the stable CRISPR mutant with the original \u003cem\u003ePUB21\u003c/em\u003e EMS mutant, a multiple sequence alignment was made of the predicted proteins (Fig. S3). The alignment revealed that both CRISPR mutant alleles had frameshift mutations and early stop codons halfway through the U-box domain, leading also to a complete loss of the ARM domain (amino acids 192\u0026ndash;364) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Meanwhile the EMS mutant contained an intact U-box domain (amino acids 22\u0026ndash;94) with an early stop codon within the ARM domain, leading to a truncated third ARM repeat and a loss of the fourth ARM repeat.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSusceptibility of\u003c/b\u003e \u003cb\u003ePUB21\u003c/b\u003e \u003cb\u003emutants to other pathogens\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate whether mutating the \u003cem\u003ePUB21\u003c/em\u003e gene influences susceptibility to other tomato pathogens, disease assays were performed on the \u003cem\u003epub21\u003c/em\u003e mutant with the necrotrophic fungus \u003cem\u003eA. solani\u003c/em\u003e. The original single \u003cem\u003epub17\u003c/em\u003e M2042 mutant (Ram\u0026iacute;rez Gaona et al, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) as well as the EMS-derived \u003cem\u003epub17\u003c/em\u003e/\u003cem\u003epub21\u003c/em\u003e double mutant showed significantly reduced lesion diameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), with the \u003cem\u003epub17\u003c/em\u003e/\u003cem\u003epub21\u003c/em\u003e double mutant showing smaller lesions than the original M2042 mutant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, \u003cem\u003epub21\u003c/em\u003e CRISPR T3 mutant families and \u003cem\u003epub21\u003c/em\u003e T3 RNAi lines were subjected to a DLA with \u003cem\u003eA. solani\u003c/em\u003e. Homozygous mutant T3 plants of CRISPR families TV202285 (TV85M), TV202269 (TV69) and TV202281 (TV81M) clearly developed significantly smaller lesions than the negative controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The RNAi T3 families TV202218, TV202234 and TV202241 displayed significantly smaller lesions compared to the negative controls, MM and T3 family TV202240, while these did not show any significant difference among each other (Fig. S4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen tested with the obligate biotrophic tomato powdery mildew pathogen \u003cem\u003ePseudoidium neolycopersici\u003c/em\u003e, plants of both the original \u003cem\u003epub17\u003c/em\u003e mutant M2042 and \u003cem\u003epub21\u003c/em\u003e CRISPR T3 family TV202269 were as susceptible as the control plants of MT or MM (Fig. S5).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cb\u003eTomato genes\u003c/b\u003e \u003cb\u003ePUB17\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ePUB21\u003c/b\u003e \u003cb\u003eare susceptibility genes towards necrotrophic fungi\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this study we report that tomato \u003cem\u003ePUB21\u003c/em\u003e, a U-box E3 ubiquitin ligase (Azevedo et al, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), acts as a S-gene during infection with the necrotrophic fungi \u003cem\u003eB. cinerea\u003c/em\u003e and \u003cem\u003eA. solani\u003c/em\u003e. In Arabidopsis, 64 \u003cem\u003ePUB\u003c/em\u003e genes have been described (Andersen et al, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) and categorized into 7 classes based on the presence/absence of domains other than the highly conserved U-box domain, such as Armadillo repeats (ARM) (Trenner et al, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). PUB21 belongs to Class IV with the presence of ARM repeats located at the C-terminus.\u003c/p\u003e \u003cp\u003eM4 progeny of EMS mutant family M2042-1-3, which showed stronger resistance against \u003cem\u003eB. cinerea\u003c/em\u003e than the original mutant, contained a mutated \u003cem\u003ePUB21\u003c/em\u003e allele in addition to the mutation in \u003cem\u003ePUB17\u003c/em\u003e. The reduced susceptibility observed in the individual F3 and F4 \u003cem\u003epub21\u003c/em\u003e mutants was comparable to that observed in the F4 and F5 \u003cem\u003epub17\u003c/em\u003e. The role of \u003cem\u003ePUB21\u003c/em\u003e in conferring susceptibility to \u003cem\u003eB. cinerea\u003c/em\u003e, independent of \u003cem\u003ePUB17\u003c/em\u003e, was confirmed through CRISPR mutation and RNAi silencing of the wild-type allele in MM background. Meanwhile, the \u003cem\u003epub17/pub21\u003c/em\u003e double mutants showed even lower susceptibility to \u003cem\u003eB. cinerea.\u003c/em\u003e These results demonstrate that mutations in \u003cem\u003ePUB17\u003c/em\u003e and \u003cem\u003ePUB21\u003c/em\u003e have an additive effect on \u003cem\u003eBotrytis\u003c/em\u003e resistance. In addition, similar to the \u003cem\u003epub17\u003c/em\u003e mutants, tomato \u003cem\u003epub21\u003c/em\u003e mutants also showed lower susceptibility to the necrotroph \u003cem\u003eA. solani\u003c/em\u003e. The \u003cem\u003epub21\u003c/em\u003e mutation in the EMS mutant resulted in the loss of the two C-terminal Armadillo repeats while the CRISPR-induced mutations resulted in a truncated U-box domain along with the complete loss of the ARM domain. The U-box is a highly conserved domain known mainly for its involvement in pairing with specific E2 proteins providing a certain level of target specificity (Trenner et al, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Mutations in the U-box domain have been reported to cause a loss of E3 activity (Turek et al, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). On the other hand, the ARM repeats are involved in protein-protein interactions for plant development, morphogenesis and hormone signaling (Coates, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Specifically, ARM repeats present in PUB proteins provide a secondary level of specificity when it comes to interacting with E2 ubiquitin-conjugating enzymes, as E2-E3 pairing is determined by both the U-box and the ARM domain (Samuel et al, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Turek et al, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Therefore, loss of these domains would hinder the ubiquitination process as specific binding of proteins targeted by PUB21 is lost. While the CRISPR mutants have a truncated U-box domain and a complete loss of the ARM repeats, two essential domains for proper E3 ligase activity, the reduced susceptibility is similar to that of the EMS mutant. This suggests that the loss of the ARM repeats is enough to lead to lower susceptibility.\u003c/p\u003e\n\u003ch3\u003ePUB21 and its close homolog PUB20 act as regulators of immunity\u003c/h3\u003e\n\u003cp\u003eStudies on the function of PUB21 in tomato or PUB21 orthologs in other plant species are scarce. Comparison of homologs may provide valuable information on the evolutionary relationship between species and insights on conserved biological pathways. In Arabidopsis, PUB21 is also known as CMPG5. The closest homolog to PUB21 in Arabidopsis is PUB20/CMPG1 (Fig. S2). Yee and Goring (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) performed a microarray study in Arabidopsis measuring transcript level changes after pathogen-derived elicitor treatment. \u003cem\u003eAtPUB21\u003c/em\u003e was induced by treatment with various pathogens, including \u003cem\u003eB. cinerea\u003c/em\u003e, \u003cem\u003ePhytophthora infestans\u003c/em\u003e, \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. \u003cem\u003ephaseolicola\u003c/em\u003e, as well as several PAMP (pathogen-associated molecular pattern) signals (Yee \u0026amp; Goring, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Given the response to pathogens and pathogen-derived molecules, AtPUB21 was suggested to be a regulator of immunity. The different time points of induction in response to different pathogen inoculations suggests that this regulatory function of AtPUB21 depends on the stage of infection. A recent article by Yi et al (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) showed that both AtPUB20 and AtPUB21 act as negative regulators of immunity at the early stage after \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. \u003cem\u003etomato\u003c/em\u003e DC3000 invasion. Both \u003cem\u003epub20\u003c/em\u003e and \u003cem\u003epub21\u003c/em\u003e mutants showed increased resistance to this bacterial pathogen.\u003c/p\u003e \u003cp\u003eIn soybean (\u003cem\u003eGlycine max\u003c/em\u003e) a \u003cem\u003ePUB20\u003c/em\u003e gene (\u003cem\u003eGlyma.14G212200\u003c/em\u003e) and a \u003cem\u003ePUB21\u003c/em\u003e-like gene (\u003cem\u003eGlyma.18G042100\u003c/em\u003e; Fig. S2) were among the highest upregulated genes after inoculation with the necrotrophic fungus \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e (Wei et al, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). On the other hand, \u003cem\u003eNicotiana benthamiana\u003c/em\u003e CMPG1 and the \u003cem\u003eN. tabacum\u003c/em\u003e ortholog ACRE74 have been reported as a positive regulators of immunity against \u003cem\u003eCladosporium fulvum\u003c/em\u003e based on the observation that RNAi-silenced plants exhibited reduced cell death induction triggered after recognition of \u003cem\u003eC. fulvum\u003c/em\u003e effector Avr9 by resistance gene \u003cem\u003eCf-9\u003c/em\u003e (Gonz\u0026aacute;lez-Lamothe et al, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe phylogenetic tree (Fig. S2) shows that the PUB20 and PUB21 proteins from Solanaceous species form two separate clusters, while AtPUB20 and AtPUB21 are in their own subgroup, suggesting that homology between the tomato and Arabidopsis \u003cem\u003ePUB21\u003c/em\u003e genes is not enough to infer a function from one species to the other. In the case of \u003cem\u003eA. thaliana\u003c/em\u003e, the amino acid sequences of AtPUB20 and AtPUB21 display 56% identity and 72% similarity. Importantly, they do not interact with the same targets, as AtPUB20 interacts with AGB1 (GTP-binding protein beta 1), while AtPUB21 does not (Kobayashi et al, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). AGB1 is reported to be required for resistance against the necrotrophic fungus \u003cem\u003ePlectosphaerella cucumerina\u003c/em\u003e (Delgado-Cerezo et al, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Evolutionary pressures may have prompted PUB gene E3 ligase diversification to manage different biotic or abiotic stresses.\u003c/p\u003e\n\u003ch3\u003eRole of tomato PUB21 in programmed cell death\u003c/h3\u003e\n\u003cp\u003eAs the loss of function of tomato \u003cem\u003ePUB21\u003c/em\u003e leads to lower susceptibility to necrotrophic fungi, we can deduce that SlPUB21 functions as a negative regulator of immunity for this class of pathogens. The presence of spontaneous necrotic spots in older leaves of the \u003cem\u003epub21\u003c/em\u003e mutants suggest a regulatory function in programmed cell death (PCD). PCD plays a pivotal role in enabling a successful infection of necrotrophic pathogens. Two types of PCD can be distinguished: apoptosis and autophagy. While the activation of either pathway leads to the appearance of necrotic plant tissue, the final outcome of each pathway leads to different effects on plant health. Research on the necrotrophic fungi \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e and \u003cem\u003eB. cinerea\u003c/em\u003e has shown that infection benefited from apoptotic PCD triggered upon inoculation with the pathogen (Dickman et al, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Veloso \u0026amp; van Kan (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) proposed that necrotrophic pathogens have the ability to temporarily suppress autophagic cell death in the early phases of infection in order to proliferate and infect the host plant. It remains to be determined which host target genes are involved in PCD regulation (Bi et al, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Leisen et al, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe infer that \u003cem\u003ePUB21\u003c/em\u003e is one of those host genes. In the \u003cem\u003epub21\u003c/em\u003e mutant the balance is shifted towards autophagic PCD. PUB21 may either positively regulate apoptosis or negatively regulate autophagy. We have shown that loss of ARM repeats of PUB21 is sufficient to reduce susceptibility to necrotrophs. Although the specific targets of PUB21 are unknown, studying the protein-protein interactions might reveal the target protein(s) of PUB21 and thereby explain how it contributes to PCD.\u003c/p\u003e\n\u003ch3\u003ePUB17 and PUB21 are involved in different PCD pathways\u003c/h3\u003e\n\u003cp\u003eAn increased resistance against both \u003cem\u003eBotrytis\u003c/em\u003e and \u003cem\u003eAlternaria\u003c/em\u003e was observed in the \u003cem\u003epub17\u003c/em\u003e/\u003cem\u003epub21\u003c/em\u003e EMS double mutant compared to the single \u003cem\u003epub17\u003c/em\u003e or \u003cem\u003epub21\u003c/em\u003e mutants. Therefore, the double mutant shows an additive effect, suggesting that each gene targets a different part of the complex cell death process. If both genes would be involved in the same pathway we would expect similar lesion diameters when comparing the double mutant with the single mutants. However, the loss of two genes involved in different aspects of the regulation of PCD, leading to stronger resistance against the necrotrophic pathogens, is accompanied by stronger pleiotropic effects on development/growth of the plant. Further testing of the double mutant and the single mutants is thus required to elucidate the roles of both PUB17 and PUB21 in PCD.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur results show that \u003cem\u003eSlPUB21\u003c/em\u003e is a key player in the susceptibility of tomato plants to the necrotrophic pathogens \u003cem\u003eB. cinerea\u003c/em\u003e and \u003cem\u003eA. solani\u003c/em\u003e. The mutation of \u003cem\u003eSlPUB21\u003c/em\u003e provides broad-spectrum resistance to these pathogens without displaying severe pleiotropic effects. The double mutant of \u003cem\u003epub17/pub21\u003c/em\u003e in tomato displayed a higher level of resistance, however, it also exhibited reduced plant size. We surmise that SlPUB21 is involved in programmed cell death regulation in a different way than SlPUB17.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePlant material\u003c/h2\u003e \u003cp\u003eTwo different tomato cultivars were used for this study: cv Micro-Tom (MT) and cv. Moneymaker (MM). MT seeds were obtained from Beekenkamp Plants B.V. (Maasdijk, The Netherlands). MT was chosen to generate an EMS population (Yan et al, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) because of its small size, short life cycle and the possibility to grow at high density (Meissner et al, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Full details on the development of the MT-EMS population have been described by Ram\u0026iacute;rez Gaona et al (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDisease assays\u003c/h2\u003e \u003cp\u003eDetached leaf assays (DLA) for both \u003cem\u003eB. cinerea\u003c/em\u003e strain B05.10 and \u003cem\u003eA. solani\u003c/em\u003e isolate CBS 143772 were performed following the methods described by Ram\u0026iacute;rez Gaona et al (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The powdery mildew disease assay with \u003cem\u003ePseudoidium neolycopersici\u003c/em\u003e isolate On-Ne was performed by spraying whole plants with a conidiospore suspension of 2.5 x 10\u003csup\u003e4\u003c/sup\u003e spores per ml, as described by Bai et al (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of mutant gene by Bulked Segregant Analysis combined with Whole Genome Sequencing (BSA-WGS)\u003c/h2\u003e \u003cp\u003eEMS mutant M2042 (Ram\u0026iacute;rez Gaona et al, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) showing reduced susceptibility to \u003cem\u003eB. cinerea\u003c/em\u003e was selfed until M4 plants were obtained (Fig. S1). Individual M4 plants were selfed for M5 seed production (M2042-1-1-17 and M2042-1-2-12) and/or crossed with MM to develop F1 seeds (M2042-1-2-12, M2042-1-3-10). F1 plants were selfed and F2 seeds were collected. F2 plants were tested for susceptibility to \u003cem\u003eB. cinerea\u003c/em\u003e by DLA to build two pools, consisting of resistant and susceptible F2 plants. Mutants obtained from the F2 generation were selfed to establish F3 lines, and subsequently F4 and F5 lines (Fig. S1a). DNA of the resistant and susceptible F2 plants was isolated following the method described by Ram\u0026iacute;rez Gaona et al (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and subsequently pooled equimolarly, resulting in two DNA pools M2042-3R and M2042-3S. Two additional pools had been made previously from the F2 progeny of M2042-1-2-12, M2042S (susceptible pool) and M2042R (intermediate resistant pool) (Ram\u0026iacute;rez Gaona et al, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e); together with a pool of wild-type Micro-Tom plants (MTWT) these were used as controls for the \u003cem\u003ePUB17\u003c/em\u003e mutation. Whole genome sequencing of the DNA pools was done as outlined in Ram\u0026iacute;rez Gaona et al (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). SNP detection was performed using SAMtools on the reads that were mapped to the tomato Heinz reference genome (version SL2.50).\u003c/p\u003e \u003cp\u003eFor each SNP the number of reads containing the reference allele (Heinz) and the number of reads with the alternative allele for each pool were recorded. Subsequent calculations and SNP filtering were performed as described by Ram\u0026iacute;rez Gaona et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Each chromosome was inspected for the presence of large difference of percentage of alternative allele per SNP position between M2042-3R and M2042-3S (∆SNP = %ALT[R] - %ALT[S]\u0026thinsp;\u0026gt;\u0026thinsp;50). On the short arm of chromosome 11 (SL2.50ch11:841716..840424), a large ∆SNP was observed between pools M2042-3R and M2042-3S. The next filtering consisted of selection of SNP positions in exons of annotated genes for which the alternative allele was present in the pools M2042-3R and M2042-3S of the extreme resistant M2042-1-3-10 but not in the MTWT pool, nor in the previously obtained resistant and susceptible pools (M2042R and M2042S) used to identify \u003cem\u003ePUB17\u003c/em\u003e (Ram\u0026iacute;rez Gaona et al, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGene expression level by RT-qPCR\u003c/h2\u003e \u003cp\u003eGene expression levels were determined by performing a RT-qPCR on plant cDNA synthesized using an iScript cDNA Synthesis kit (BioRad) on RNA extracted through an RNeasy Plant Mini Kit (Qiagen). Specific primers were developed for each gene (Table S1). Elongation factor 1 alpha (\u003cem\u003eEf1α\u003c/em\u003e) was used as reference gene. RT-qPCR was performed using a CFX96 Real-Time PCR machine (BioRad) with two technical replicates used per sample. Relative expression of \u003cem\u003ePUB17\u003c/em\u003e or \u003cem\u003ePUB21\u003c/em\u003e was calculated with the ΔΔC\u003csub\u003eT\u003c/sub\u003e method (Livak \u0026amp; Schmittgen, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRNAi and CRISPR transformation for confirmation of candidate gene\u003c/h2\u003e \u003cp\u003e \u003cem\u003ePUB21\u003c/em\u003e RNAi constructs were generated using the binary vector pHellsgate8 (Helliwell and Waterhouse 2003); full details are described in Ram\u0026iacute;rez Gaona et al (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This vector contains a CaMV 35S promoter driving the expression of the inverted repeat and a kanamycin resistance gene as a selectable marker. Primers were designed for \u003cem\u003ePUB21\u003c/em\u003e to amplify fragments from tomato MM gDNA. Primer sequences are shown in Table S1. RNAi fragment 1 targets the region between the U-box domain and the Armadillo (ARM) repeats of the PUB21 protein, while the RNAi fragment 10 targets the ARM repeats domain.\u003c/p\u003e \u003cp\u003eA CRISPR/Cas9 construct was developed to create deletions within the \u003cem\u003ePUB21\u003c/em\u003e coding sequence, using 3 sgRNAs alongside the Cas9 endonuclease gene and the NPTII plant selectable marker. The sgRNAs were designed as described by Santill\u0026aacute;n Mart\u0026iacute;nez et al (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). To select the best 3 sgRNAs, in addition to sgRNA scorer (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sgrnascorer.cancer.gov\u003c/span\u003e\u003cspan address=\"https://sgrnascorer.cancer.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e [Chari et al, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e]), two additional tools were used: GPP sgRNA Designer (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design\u003c/span\u003e\u003cspan address=\"https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e [Sanson et al, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e]), and WU-CRISPR (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispr.wustl.edu\u003c/span\u003e\u003cspan address=\"http://crispr.wustl.edu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e [Wong et al, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2015\u003c/span\u003e]). The construct was assembled using a Golden Gate cloning system (Engler et al, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) with plasmids from Addgene as described by Santill\u0026aacute;n Mart\u0026iacute;nez et al (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The plasmids were cloned using \u003cem\u003eE.coli\u003c/em\u003e DH5α.\u003c/p\u003e \u003cp\u003eThe two RNAi constructs and one CRISPR/Cas9 construct for \u003cem\u003ePUB21\u003c/em\u003e were transformed into electrocompetent \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e AGL1\u0026thinsp;+\u0026thinsp;virG cells. Transformation of tomato cv. MM was carried out as previously described (Huibers et al, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). DNA was isolated from young leaves using CTAB buffer (1 M Tris-HCl pH 7.5, 0.5 M EDTA pH 8.0, 5 M NaCl, 2% CTAB) essentially as described by Doyle (1991). To confirm the integration of the T-DNAs of the silencing constructs in the genome of the RNAi transformants, a PCR was performed to detect the presence of the \u003cem\u003eNPTII\u003c/em\u003e gene and 35S promoter using DreamTaq DNA polymerase (Thermo Scientific, Bleiswijk, The Netherlands). Primer sequences are shown in Table S1. To determine the presence of mutations in the CRISPR transformants PCRs were performed using primers flanking the sgRNA target sites. The PCR products were sent for sequencing to Macrogen Europe (Amsterdam, The Netherlands).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData points for each DLA experiment were subjected to an ANOVA F-test using R studio v 1.1.463 (Allaire, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The ANOVA test was followed by a Post Hoc test using Tukey HSD method to perform multiple pairwise-comparisons. Differences were considered significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eProtein alignment and prediction tools\u003c/h2\u003e \u003cp\u003eSequencing of the \u003cem\u003ePUB21\u003c/em\u003e EMS mutant and CRISPR T3 mutants was performed using primers covering the complete genomic sequence (Table S1). MM and MT DNA samples were taken along as controls. The \u003cem\u003ePUB21\u003c/em\u003e sequences were assembled by aligning the DNA fragments using the plasmid editor ApE (Davis \u0026amp; Jorgensen, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Each complete DNA sequence was translated to RNA using the ExPASy translate tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/translate/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/translate/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e [Gasteiger et al, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2003\u003c/span\u003e]). Protein alignment was done using the multiple sequences alignment program Clustal Omega provided by EMBL-EBI (Madeira et al, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Protein domains were predicted using the ScanProsite tool (De Castro et al, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The Arabidopsis PUB protein sequences were obtained from TAIR (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e [Rhee et al, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2003\u003c/span\u003e]), while the rest of the PUB protein sequences were obtained from NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). After protein alignment, MAFFT7 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://mafft.cbrc.jp/alignment/server/\u003c/span\u003e\u003cspan address=\"https://mafft.cbrc.jp/alignment/server/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ) was used to build the phylogenetic tree.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Fien Meijer-Dekens and Bertus van der Laan for taking care of the plants in the greenhouse, and Jaap Wolters for providing inoculum of \u003cem\u003eAlternaria solani\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYB, AMAW, JALvK and RGFV conceived the study. AvT performed the screening of the EMS population. MRG, DS and AvT performed the experiments. JALvK provided disease testing resources and protocols. MRG, AMAW and YB analysed the results. MRG wrote the manuscript with input from all co-authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was financially supported by grants from Foundation Topconsortium voor Kennis en Innovatie (TKI) Horticulture \u0026amp; Starting Materials projects EZ-2012-07 and TU-18015.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated and analyzed supporting the findings of the current work are available within the manuscript and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\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\n\u003cli\u003eAllaire J. 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Phytopathology 2022;112:1739-1752.\u003c/li\u003e\n\u003cli\u003eWilliamson B, Tudzynski B, Tudzynski P, van Kan JAL. \u003cem\u003eBotrytis cinerea\u003c/em\u003e: the cause of grey mould disease. Mol. Plant Pathol. 2007;8:561-580.\u003c/li\u003e\n\u003cli\u003eWong N, Liu W, Wang X. WU-CRISPR: characteristics of functional guide RNAs for the CRISPR/Cas9 system. Genome Biol. 2015;16:218.\u003c/li\u003e\n\u003cli\u003eYan Z, Appiano M, van Tuinen A, Meijer-Dekens F, Schipper D, Gao D, Huibers R, Visser RGF, Bai Y, Wolters A-MA. Discovery and characterization of a novel tomato \u003cem\u003emlo\u003c/em\u003e mutant from an EMS mutagenized Micro-Tom population. Genes\u003cem\u003e \u003c/em\u003e2021;12:719.\u003c/li\u003e\n\u003cli\u003eYee D, Goring DR. The diversity of plant U-box E3 ubiquitin ligases: from upstream activators to downstream target substrates. J. Exp. Bot. 2009;60:1109-1121.\u003c/li\u003e\n\u003cli\u003eYi SY, Nekrasov V, Ichimura K, Kang SY, Shirasu K. Plant U-box E3 ligases PUB20 and PUB21 negatively regulate pattern-triggered immunity in Arabidopsis. Plant Molecular Biology 2024;114:7.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Tomato, EMS, PUB21, Susceptibility gene (S-gene), Botrytis cinerea, Alternaria solani","lastPublishedDoi":"10.21203/rs.3.rs-6473041/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6473041/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCultivated tomato is susceptible to necrotrophic pathogens \u003cem\u003eBotrytis cinerea\u003c/em\u003e and \u003cem\u003eAlternaria solani\u003c/em\u003e. No dominant resistance against these pathogens has been reported in wild relatives of tomato.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThrough screening of a tomato Micro-Tom EMS population we identified a mutant that showed decreased susceptibility to both necrotrophic fungi. Previously, we reported a mutation in the tomato \u003cem\u003ePUB17\u003c/em\u003e gene as the cause of reduced susceptibility in this mutant. Surprisingly, M4 progeny of one M3 plant homozygous for the \u003cem\u003epub17\u003c/em\u003e mutation showed segregation with some plants displaying an even higher level of resistance than the \u003cem\u003epub17\u003c/em\u003e mutant. This highly resistant progeny was shown to contain a mutation in tomato \u003cem\u003ePUB21\u003c/em\u003e in addition to the mutation in \u003cem\u003ePUB17\u003c/em\u003e. The role of \u003cem\u003ePUB21\u003c/em\u003e as a susceptibility factor for both necrotrophic fungi was confirmed in RNAi-silenced and CRISPR-mutated transformants.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIn this study we identified a new \u003cem\u003ePUB\u003c/em\u003e gene, \u003cem\u003eSlPUB21\u003c/em\u003e, involved in susceptibility of tomato to necrotrophic pathogens. We showed that mutation of this gene resulted in increased resistance against these pathogens.\u003c/p\u003e","manuscriptTitle":"Mutation of PUB21 in tomato leads to reduced susceptibility to necrotrophic fungi","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-02 11:31:16","doi":"10.21203/rs.3.rs-6473041/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-26T11:00:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-20T02:40:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"265804823862152864549244728763059912616","date":"2025-05-15T07:26:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-02T14:26:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"249510783244778844803131989773167401103","date":"2025-04-28T10:22:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-28T09:50:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-28T09:40:01+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-28T09:11:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-28T09:01:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-04-28T09:00:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1f20e7af-0552-497b-bc72-96e2a46b09bd","owner":[],"postedDate":"May 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-22T06:38:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-02 11:31:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6473041","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6473041","identity":"rs-6473041","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}