Mutation of PUB17 in tomato leads to reduced susceptibility to necrotrophic fungi

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.