CBL10 was shown to be a key gene for salt tolerance in Arabidopsis thaliana. In this study, we evaluated the role of CBL10 in the tobacco salt tolerance response by characterizing the gene editing-induced loss-of-function knockout mutants of the two NtCBL10 homeologous genes NtCBL10A and NtCBL10B. The importance of NtCBL10 for the response to salinity was evidenced by the salt supersensitivity of the Nt-cbl10a10b double mutants, with fast-developing chlorosis and severe necrotic lesions on leaves. Stomatal conductance and photochemical efficiency of photosystem 2 (PhiPS2) of the Nt-cbl10a10b double mutant were significantly inhibited already at a very early stage of the salt stress response. Leaf Na+ concentrations were not much affected in these plants, but the Cl- content of the Nt-cbl10a10b double mutants was significantly lower than that of wild-type plants, which is the first report of CBL10 in the regulation of Cl- homeostasis. Interestingly, the necrosis phenotype of Nt-cbl10a10b double mutants was dependent on light, while the chlorosis phenotype of Nt-cbl10a10b double mutants was light-independent. Different from the previous studies that focus on the role of CBL10 in Na+ homeostasis regulation, this study indicates that NtCBL10 is a key component in regulating multiple aspects of ion homeostasis under salt stress.

Comparative transcriptomics has identified several candidate genes contributing to the resistance of Cucumis metuliferus against Meloidogyne incognita. The Southern root-knot nematode (Meloidogyne incognita) is a significant threat to Cucurbitaceae crops. The African horned melon (Cucumis metuliferus), a wild relative, exhibits high resistance to this nematode. To explore the resistance mechanism, phenotypic analyses were conducted on a susceptible inbred line (CM27) and a resistant inbred line (CM3). CM3 exhibited enhanced root biomass and significantly higher resistance compared to CM27, with poor nematode development observed in CM3 roots. Transcriptomic profiling at multiple post-infection time points revealed 2243 and 3700 differentially expressed genes (DEGs) in CM3 and CM27, respectively. Among these, the top ten DEGs upregulated exclusively in CM3 were functionally analyzed using virus-induced gene silencing (VIGS). Silencing of EVM0019904 or EVM0017058 in CM3 led to susceptibility to M. incognita. These findings provide novel insights into the resistance mechanisms of M. incognita in C. metuliferus and offer potential resources for breeding nematode-resistant Cucurbitaceae crops.

The infection of plants by pathogens is an intricate process in which genes from both the host and pathogen contribute to the infection process. Susceptibility (S) genes have been defined as plant genes that encode functions that are exploited by pathogens to invade and reproduce in host plants. Mutations in S-genes therefore result in reduced susceptibility to the pathogen. The identity and mode of action of S-genes in interactions of plants with biotrophic fungi have been studied since the cloning of the barley mlo gene in the late 1990s. The infection strategy of necrotrophic fungi, however, substantially differs from that of biotrophic fungi and therefore is likely to be facilitated by distinct physiological processes in a host plant. There is a rapidly increasing amount of information about S-genes in plants that facilitate the infection by necrotrophic fungi and their mode of action. In this review, we summarise the current knowledge on plant S-genes for susceptibility to necrotrophic fungi and categorise them based on cellular compartments and physiological processes. With recent examples, we subsequently discuss the challenges and opportunities of exploiting impaired plant S-genes for breeding crops with disease resistance.

Genetic resistance is an important mitigation strategy in commercial tomato cultivation against leaf mold disease caused by the fungal pathogen Fulvia fulva . In recent years, the widely deployed Cf-9 resistance locus has been overcome, which has prompted breeders to deploy the novel resistance locus Cf-6 . However, Cf-6 -mediated resistance has already been overcome in several locations worldwide. To date, the identity of Cf-6 , as well as the gene encoding the matching Avr6 effector from F. fulva have remained unknown. Here, we report the identification of Avr6 using a comparative genomics strategy. Whole genome sequencing of Cf-6 -breaking F. fulva strains revealed that the gene coding for the known secreted effector Ecp5 is either deleted or mutated in these strains. Ecp5 was subsequently confirmed to be Avr6 through potato virus X-mediated expression in Cf-6 tomato plants, as well as by gene knockout and complementation experiments. Using the Avr6-triggered hypersensitive response as a marker for disease resistance, we mapped the Cf-6 locus to the short arm of chromosome 12 in tomato. In addition to identifying Avr6 and setting the foundation for cloning of Cf-6 , this work highlights the urgent need for more research into durable gene-for-gene resistance in tomato against F. fulva . ### Competing Interest Statement The authors have declared no competing interest. European Union Horizon 2020 HARNESSTOM project, 101000716

Cyclic nucleotide-gated ion channel (CNGC) genes play vital roles in plant growth, development, and responses to both biotic and abiotic stresses. However, the current research on CNGCs in potato (Solanum tuberosum) remain largely uncharacterized. Blackleg disease is one of the most devastating diseases worldwide, causing severe yield losses. Understanding the role of the StCNGC gene family in blackleg resistance is therefore of significant importance. In this study, we identified 11 StCNGC genes in the potato genome and conducted phylogenetic analysis, gene structure characterization, and conserved motif prediction. Expression patterns were examined in different tissues and under stress conditions. The identified StCNGCs were classified into five groups, and showed conserved gene structures and motifs within groups. Most StCNGCs were induced under biotic stress conditions. Notably, silencing StCNGC2 conferred resistance to blackleg disease and resulted in the upregulation the pathogenesis-related marker gene StPR1. Together, these findings suggest that StCNGC2 plays a crucial role in potato defense against blackleg disease and provide a foundation for further functional studies of the StCNGC gene family.

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. 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. 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.

Rice straw returned directly to the field offers various benefits, including improved soil structure, water conservation, and crop growth. However, straw decomposition can create unfavorable conditions, such as soil acidification and gleyization, that negatively impact rice growth. This study conducted three experiments to assess the effects of returning straw to double-cropping rice fields by screening for decomposing bacteria, nutrient release, and complete straw return. The results from these experiments indicated that the decomposition efficiency of straw significantly increased when both pig manure and straw were returned to the field simultaneously. Additionally, the introduction of exogenous calcium oxide increased the soil pH by more than 0.3, effectively preventing soil acidification. Furthermore, after four years of continuous straw return, the addition of calcium oxide or pig manure significantly increased the annual rice yield. These findings provide scientific evidence supporting the long-term advantages of incorporating straw return into the soil fertilization practices of double-cropping rice systems, highlighting the potential for sustained improvement in soil fertility.

Cucumber production is seriously constrained by Meloidogyne incognita. Because no resistance resources to the pathogen have been reported, disabling susceptibility genes may represent a novel breeding strategy to introduce resistance against this nematode in cucumber. Here, we studied the clade V MLO genes for their involvement in the interaction between cucumber and M. incognita. Our results showed that Arabidopsis clade V MLO mutants were resistant to M. incognita. Cucumber has three clade V MLO genes, CsaMLO1, CsaMLO8 and CsaMLO11, with upregulated expression upon inoculation with M. incognita. Heterologous overexpression of CsaMLO1, CsaMLO8 and CsaMLO11 in Arabidopsis mutants restored susceptibility to varying degrees. Silencing and knockout of individual clade V MLO genes in cucumber reduced susceptibility to M. incognita. The cucumber CRISPR mutants produced similar fruits as the wild type (WT) did. Although the yields of two single mutants (M111 and M112) and two double mutants (M81 M111 and M81 M112) were reduced compared to WT, the yields of M81 and M82 were not decreased. In summary, clade V MLO genes function as susceptibility genes for M. incognita in cucumber. Among them, CsaMLO8 may be the most promising candidate for M. incognita resistance breeding in cucumber.

Key message The tomato Ty-6 gene conferring resistance against begomoviruses has been cloned and shown to be a variant of DNA polymerase delta subunit 1. Abstract Ty-6 is a major resistance gene of tomato that provides resistance against monopartite and bipartite begomoviruses. The locus was previously mapped on chromosome 10, and in this study, we fine-mapped Ty-6 to a region of 47 kb, including four annotated candidate genes. Via whole-genome resequencing of Ty-6 breeding lines and several susceptible breeding lines, the polymorphisms in gene sequences were discovered and gene-associated markers were developed for marker-assistant breeding. Further, virus-induced gene silencing and candidate gene overexpressing in susceptible tomatoes revealed that Ty-6- mediated resistance is controlled by Solyc10g081250, encoding the DNA polymerase delta subunit 1, SlPOLD1 . The single nucleotide polymorphism of Ty-6 results in an amino acid change that might influence the fidelity of virus DNA replication.

Applying organic fertilizer is an important traditional agriculture practice in China. With the development of its fertilizer industry, the fertilizer application rate in China has increased annually. However, a large amount of organic matter, such as straw, cannot be fully utilized. The aim of this study was to analyze the effects of five rice straw return methods on soil fertility and rice yield in double-cropping rice areas through long-term cultivation experiments. Straw return increases the contents of soil nutrients, especially, potassium, and activities of enzymes associated with the C- and N-cycle. The treatment with early rice returning + early rice straw full crushing and returning + late rice straw full mulching and returning + deep rotary tillage or deep tillage (depth, approximately 0-20 cm) exhibited the best effects, which increased the yield of early and late rice by more than 5 %. These results provide a robust theoretical basis for improving the soil fertility and rice yield in doublecropping rice areas using straw return methods.

Soil organic carbon (SOC) plays a crucial role in plant nutrient supply and soil physical, chemical, and biological function regulation. Factors such as climate change, human activities, and farm management practices can adversely affect SOC. Here, paddy soil in the double-cropping rice area of Hunan Province of China was cultured with eight kinds of organic materials, and the effects of organic fertilization on SOC content, humus content, and soil carbon sequestration efficiency were analyzed. The addition of straw, green fertilizer, and organic fertilizer had a positive influence on active SOC content. Straw addition had the most prolonged impact on soil microbial biomass carbon content, and green manure had the most rapid influence on soil dissolved organic carbon content. All organic materials enhanced the soil humus content; furthermore, the addition of organic fertilizers significantly improved the carbon sequestration efficiency among all treatments. The results of controlled culture experiments in paddy soils established that applying organic materials can increase SOC content, active carbon components, soil humus content, and the carbon fixation rate, thereby improving soil fertility. This study provides a theoretical basis for the optimal fertilization of paddy soil with organic material addition in China.

The Tm-1 allele from Solanum pennellii accessions together with an additional, likely recessive, locus are required for complete ToBRFV resistance. The Tobamovirus Tomato Brown Rugose Fruit Virus (ToBRFV) poses a significant threat to global tomato production. ToBRFV is a mechanically transmitted virus containing a single-stranded positive sense RNA genome. Disease symptoms include brown, rough patches on fruit surfaces, leaf mosaicism and shape abnormalities, and, in advanced stages, total collapse of infected plants. ToBRFV was first detected in the Middle East in 2014 and has rapidly spread to multiple countries across Asia, Europe, and America. In recent years, numerous studies have focused on the identification of ToBRFV resistance traits that are suitable for tomato breeding programs. In this study, we identified five ToBRFV-resistant accessions of Solanum pennellii, a wild relative of cultivated tomato. We confirmed that the major gene controlling this resistance trait is the S. pennellii allele of Tm-1. Tm-1 was previously identified in S. habrochaites as a semidominant Tomato Mosaic Virus (ToMV) resistance gene. Our results show that full resistance to ToBRFV disease requires an additional undescribed locus. These results show the potential of S. pennellii as a novel source of resistance against ToBRFV.

Powdery mildew (PM), triggered by Oidium neolycopersici, represents a significant threat and a major concern for the productivity of tomato plants (Solanum lycopersicum L.). The presence of susceptibility (S) genes in plants facilitates pathogen proliferation and their dysfunction can lead to a recessively inherited broad-spectrum and durable type of resistance. Past studies have demonstrated that disrupting the function of DND1 (Defense No Death 1) increases plant resilience against various pathogens, such as powdery mildew (PM), but this comes at the cost of negatively affecting the overall health and vigor of the plant. To investigate the possibility of minimizing the adverse effects of the dnd1 mutation while boosting disease resistance, a CRISPR-Cas9 construct with four single guide RNAs targeting three exons of SlDND1 (Solyc02g088560.4.1) was designed and introduced into the tomato variety Moneymaker (MM) through Agrobacterium tumefaciens-mediated transformation. Three T1 lines (named E1, E3 and E4) were crossed with MM and then selfed to produce TF2 families. All the TF2 plants in homozygous state dnd1/dnd1, showed reduced PM symptoms compared to the heterozygous (DND1/dnd1) and wild type (DND1/DND1) ones. Two full knock-out (KO) mutant events (E1 and E4) encoding truncated DND1 proteins, exhibited clear dwarfness and auto-necrosis phenotypes, while mutant event E3 harbouring deletions of 3 amino acids, showed normal growth in height with less auto-necrotic spots. Analysis of the 3D structures of both the reference and the mutant proteins revealed significant conformational alterations in the protein derived from E3, potentially impacting its function. A dnd1/dnd1 TF2 line (TV181848-9, E3) underwent whole-genome sequencing using Illumina technology, which confirmed the absence of off-target mutations in selected genomic areas. Additionally, no traces of the Cas9 gene were detected, indicating its elimination through segregation. Our findings confirm the role of DND1 as an S-gene in tomato because impairment of this gene leads to a notable reduction in susceptibility to O. neolycopersici. Moreover, we provide, for the first time, a dnd1 mutant allele (E3) that exhibits fitness advantages in comparison with previously reported dnd1 mutant alleles, indicating a possible way to breed with dnd1 mutants.

The southern root-knot nematode, Meloidogyne incognita, is a highly serious plant parasitic nematode species that causes significant economic losses in various crops, including cucumber (Cucumis sativus L.). Currently, there are no commercial cultivars available with resistance to M. incognita in cucumber. However, the African horned melon (Cucumis metuliferus Naud.), a semi-wild relative of cucumber, has shown high resistance to M. incognita. In this study, we constructed an ultrahigh-density genetic linkage bin-map using low-coverage sequences from an F2 population generated through the cross between C. metuliferus inbred lines CM3 and CM27. Finally, we identified a QTL (quantitative trait locus, QTL3.1) with a LOD (logarithm of the odds) score of 3.84, explaining 8.4% of the resistance variation. Subsequently, by combining the results of qPCR (quantitative PCR) and VIGS (virus-induced gene silencing), we identified two genes, EVM0025394 and EVM0006042, that are potentially involved in the resistance to M. incognita in CM3. The identification of QTLs and candidate genes in this study serve as a basis for further functional analysis and lay the groundwork for harnessing this resistance trait.

Increasing natural resistance and resilience in plants is key for ensuring food security within a changing climate. Breeders improve these traits by crossing cultivars with their wild relatives and introgressing specific alleles through meiotic recombination. However, some genomic regions are devoid of recombination especially in crosses between divergent genomes, limiting the combinations of desirable alleles. Here, we used pooled-pollen sequencing to build a map of recombinant and non-recombinant regions between tomato and five wild relatives commonly used for introgressive tomato breeding. We detected hybrid-specific recombination coldspots that underscore the role of structural variations in modifying recombination patterns and maintaining genetic linkage in interspecific crosses. Crossover regions and coldspots show strong association with specific TE superfamilies exhibiting differentially accessible chromatin between somatic and meiotic cells. About two-thirds of the genome are conserved coldspots, located mostly in the pericentromeres and enriched with retrotransposons. The coldspots also harbor genes associated with agronomic traits and stress resistance, revealing undesired consequences of linkage drag and possible barriers to breeding. We presented examples of linkage drag that can potentially be resolved by pairing tomato with other wild species. Overall, this catalogue will help breeders better understand crossover localization and make informed decisions on generating new tomato varieties.

The CALCINEURIN B-LIKE PROTEIN family genes (CBLs) encode a group of plant-specific calcium sensor proteins that play critical roles in plant development and stress response. Although genome-wide analysis of CBL family genes has been conducted in several diploid plant species, less is known about their functions in allopolyploid plant species. This study conducted a detailed analysis of tobacco CBL family genes. Of the 24 NtCBL genes identified, 14 were derived from the maternal genome donor N. sylvestris, and 10 were from the paternal genome donor N. tomentosiformis. Phylogenetic analysis revealed that NtCBLs could be clustered into three different groups with distinct N-terminal characteristics. All NtCBLs contain EF-hand motifs, but amino acid substitutions and configuration changes may be responsible for variations in their Ca2+-binding abilities. Alternative splicing is common for NtCBL gene transcripts, which may contribute to variations in translation efficiency or protein interaction preferences. Several NtCBL genes exhibited shoot-or root-predominant expression patterns, and some were responsive to salt and/or drought stress. NtCBL4A-1, an ortholog of Arabidopsis thaliana AtCBL4 (SOS3), was predominantly expressed in roots and exhibited a lower gene expression level under salt stress. Further func-tional analysis showed that overexpression of NtCBL4A-1 increased the sensitivity of transgenic tobacco to Na+- induced salt stress, which is inconsistent with the role of AtCBL4 in contributing to salt tolerance. This study provides data for identifying CBL gene functions and selecting candidate stress-tolerant genes in N. tabacum. Additionally, the results may aid gene function studies in other allopolyploid plant species.

Summary Leaf mould, caused by Fulvia fulva , is a devastating disease of tomato plants. In many commercial tomato cultivars, resistance to this disease is governed by the Cf-9 locus, which comprises five paralogous genes ( Cf-9A–9E ) that encode receptor-like proteins. Two of these proteins contribute to resistance: Cf-9C recognizes the previously identified F. fulva effector Avr9 and provides resistance during all plant growth stages, while Cf-9B recognises the yet-unidentified F. fulva effector Avr9B and provides mature plant resistance only. In recent years, F. fulva strains have emerged that have overcome the Cf-9 locus, with Cf-9C circumvented through Avr9 deletion. To understand how Cf-9B is circumvented, we set out to identify Avr9B . Comparative genomics, in planta transient expression assays and gene complementation experiments were used to identify Avr9B , while gene sequencing was used to assess Avr9B allelic variation across a worldwide strain collection. A strict correlation between Avr9 deletion and resistance-breaking mutations in Avr9B was observed in strains recently collected from Cf-9 cultivars, whereas Avr9 deletion but no mutations in Avr9B were observed in older strains. This research showcases how F. fulva has evolved to sequentially break down the two functional resistance genes of the complex Cf-9 locus and highlights that this locus now has limited value for controlling leaf mould disease in worldwide commercial tomato production.

To explore specific components of resistance against the tomato-adapted powdery mildew pathogen Pseudoidium neolycopersici (On) in the model plant Arabidopsis, we performed a disease assay in 123 accessions. When testing the resistance in the F1 from crossings between resistant accessions with susceptible Col-0 or Sha, only the progeny of the cross between accession Bla-6 and Col-0 displayed a completely resistant phenotype. The resistance in Bla-6 is known to be specific for Pseudoidium neolycopersici. QTL analysis and fine-mapping through several rounds of recombinant screenings allowed us to locate a major resistance QTL in an interval on chromosome 1, containing two candidate genes and an intergenic insertion. Via CRISPR/Cas9 targeted mutagenesis, we could show that knocking out the ZED-1 RELATED KINASE 13 (ZRK13) gene compromised the On resistance in Bla-6. Several polymorphisms are observed in the ZRK13 allelic variant of Bla-6 when compared to the Col-0 protein.

Tomato brown rugose fruit virus (ToBRFV) is a recently emerged serious viral threat to tomato production. The virus is named after its symptoms consisting of characteristic brown wrinkled (rugose) patches on the fruits of infected tomato plants. ToBRFV is a member of the genus Tobamovirus and a very stable mechanically transmitted virus. So far, most tomato cultivars are susceptible, enabling a swift spread of ToBRFV. In this review, we present strategies to halt devastating disease outbreaks of ToBRFV based on the collective research data of various tobamovirus–plant interactions. Viruses, like ToBRFV, are biotrophic pathogens with small genomes. Hence viral proliferation depends on various host factors, also termed susceptibility (S) genes. However, S genes often have an intrinsic function for the host plant. Thus, mutations in S genes may lead to pleiotropic phenotypes. Therefore, identifying mutant variants of S genes with no pleiotropic effects is essential for exploring impaired S genes in breeding tomatoes resistant to ToBRFV.

Necrotrophic fungi, such as Botrytis cinerea and Alternaria solani, cause severe damage in tomato production. There is no report of any single resistance (R) gene that can provide dominant resistance against necrotrophic fungi (Davis et al., 2009; Finkers et al., 2007). In this study, we demonstrate that breeding tomato for broad-spectrum resistance towards necrotrophic fungi can be achieved by editing host susceptibility (S) genes. Screening a tomato Micro-Tom (MT) EMS population (Yan et al., 2021) we identified mutant M2042 showing a 20%–30% reduction in lesion diameter after B. cinerea infection when compared to the wild-type MT control in a detached leaf assay (DLA). A bulk segregant analysis and whole genome sequencing (BSA-seq) approach was applied in the F2 population derived from the cross of M2042 and Moneymaker (MM, susceptible to B. cinerea; Methods S1, Figures S1 and S2). Two potential non-synonymous mutations were identified. The first mutation (A → T SNP) was at position 1477 of the coding region of gene Solyc02g072080 resulting in a premature stop codon R493* (Figure 1a). This gene is the tomato orthologue of PUB17, a conserved U-box-containing E3 ubiquitin ligase (Trenner et al., 2022). The second mutation (G → A SNP) occurred in exon 5 of gene Solyc02g078920 resulting in an amino acid change G158D. This gene encodes an S1/P1 nuclease. Of the two candidate genes, only the expression of PUB17 was altered in the mutant at 24 and 48 h after Botrytis inoculation (Figure 1b). Further fine mapping showed that the SNP in PUB17 co-segregated with the resistance (Figure 1c). To confirm that the PUB17 gene is an S gene to B. cinerea, knock-down and out transformants were produced using RNAi and CRISPR (Table S1). In both DLA and stem assays, significantly reduced susceptibility was observed in T3 well-silenced plants of three independent RNAi transformants (Figures S3 and S4) generated by transforming MM with RNAi constructs (Figure 1a). For CRISPR analysis, a construct with 4 sgRNAs targeting the different protein domains were made (Figure 1a) to transform MM. Homozygous mutant T3 progeny could be obtained from T1 transformants 7 (mutant allele 1 and 2) and 36 (mutant allele 3 and 4; Figure 1a, Figures S5 and S6). For each mutant allele, two T3 families were tested with B. cinerea in both stem assay and a DLA. All T3 families exhibited significantly lower disease severity compared with MM and CRISPR T2 family TV33 containing wild-type PUB17 alleles (Figure 1d, Figure S4). A multiple sequence alignment of the predicted PUB17 proteins (Figure S7) revealed that mutations of all the mutant alleles led to partial deletion of the Armadillo repeats (ARM; Figure 1e). Additional to B. cinerea, the pub17 mutants (both EMS and CRISPR) showed significantly reduced lesion diameters after A. solani infection, when compared to the corresponding controls (MM or MT; Figure 1f). Meanwhile, no increased susceptibility was observed in these pub17 mutants when tested with obligate biotrophic tomato powdery mildew pathogen Pseudoidium neolycopersici (Figure S8). The original EMS-mutant plant M2042 had slightly smaller leaves than wild-type MT, and the leaves were slightly wrinkled. To assess the breeding value of the EMS pub17 mutation, we introgressed the EMS-mutant allele into different tomato breeding lines (Figure S9). Results demonstrated that occurrence of autonecrosis of pub17 mutants depends on the genetic background. We succeeded in producing F1 hybrids carrying homozygous pub17 alleles with a plant phenotype and fruit setting indistinguishable from the F1 without the pub17 mutation. In this study, we report for the first time that SlPUB17 operates as an S gene during plant interaction with necrotrophic pathogens. The Arabidopsis PUB family consists of 64 PUB genes classified into seven classes and their encoded proteins contain a highly conserved U-box domain along with various additional domains (Trenner et al., 2022). PUB17 belongs to Class V containing a U-box N-terminal domain (UND) at the N-terminus and ARM repeats at the C-terminus (Figure 1a). The individual EMS- and CRISPR-induced mutations resulted in the loss of multiple ARM repeats for all mutant alleles (Figure 1e). ARM repeats participate in protein–protein interactions (Samuel et al., 2006). The loss of these repeats would hamper the interaction of proteins associated with PUB17 and consequently the ubiquitination of proteins targeted by PUB17. Previous studies on PUB17 homologues of Arabidopsis thaliana, Nicotiana tabacum, potato and cotton showed that PUB17 is a positive regulator of immunity against different diseases caused by biotrophic and hemibiotrophic pathogens, such as Cladosporium fulvum, Phytophthora infestans and Verticillium dahliae (Ni et al., 2010; Tian et al., 2007; Yang et al., 2006; Zhang et al., 2016). In contrast, our results revealed a role of PUB17 as an S gene for necrotrophic pathogens by acting as a negative regulator of immunity against B. cinerea and A. solani. Given that the SlPUB17 mutants exhibit lower susceptibility to necrotrophic pathogens manifested as smaller lesion diameters, we deduce that PUB17 operates as a positive regulator of programmed cell death (PCD). The role of PUB17 in the PCD pathway has yet to be confirmed, since the specific target(s) for degradation by the ubiquitin ligase PUB17 remain unknown. Yet, as several necrotrophic pathogens rely on (programmed) cell death to facilitate growth, it is plausible to propose that the resistance observed in PUB17 mutants to Botrytis and Alternaria could be linked to interference with PCD. Since S genes are shown to be conserved across different plant species (Koseoglu et al., 2022), we expect that the identified PUB17 gene might be explored in a range of crops for resistance to B. cinerea and A. solani by induced mutations via mutagenesis, RNAi (easy for polypoid crops) and especially gene-editing with CRISPR. 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. We thank Fien Meijer-Dekens and Bertus van der Laan for taking care of the plants in the greenhouse. Thanks also to Alejandro Thérèse Navarro for his guidance in R programming. None declared. YB, AMAW, JALvK and RGFV conceived the study. AvT performed the screening of the EMS population. MRG, DS, AvT and PJW performed the experiments. PJW and JALvK provided disease testing resources and protocols. MRG, AMAW and YB analysed the results. AK participated in advanced material production. MRG wrote the manuscript with input from all co-authors. The data used to support the findings of this study are available in the main text and Supporting information of this article. Methods S1 Materials and methods. Figure S1 Pedigree of M2042 EMS mutant. Figure S2 Results from the BSA-seq analysis. Figure S3 Analysis of PUB17 RNAi transformants. Figure S4 Botrytris cinerea stem assay results of PUB17 transformants. Figure S5 Analysis of PUB17 CRISPR transformants. Figure S6 Multiple genomic sequence alignment of Solanum lycopersicum wild-type and mutant PUB17 alleles. Figure S7 Multiple protein sequence alignment of Solanum lycopersicum wild-type and mutant PUB17 alleles. Figure S8 Powdery mildew disease scoring results of PUB17 CRISPR mutants. Figure S9 EMS-mutant pub17 pre-breeding results. Table S1 List of primers. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.