Sm is a single dominant resistance gene in tomato
Previous studies have shown that Sm is a dominant gene for gray leaf spot resistance. In this study, we chose the resistant tomato line Motelle (Mt, containing the Sm gene) and the susceptible line Moneymaker (Mm) as parents to generate an F2 population to map the Sm gene. The GLS symptoms in the resistant line Motelle and susceptible line Moneymaker were as shown in Fig. 1A. We performed a disease assay on the F2 population at 5-7 days post inoculation with S. lycopersici (Fig. 1B). In the 476 F2 population, 365 were regarded as resistant plants (DS levels between 0 and 1), and 111 were considered susceptible plants with DS levels of 2-5. The ratio of resistant plants to susceptible plants was approximately 3.29:1, which was consistent with the Mendelian segregation ratio of 3:1, as shown in Fig. 1C (χ2 = 0.6302, P > 0.05)This result suggests that the Sm gene confers resistance singly via dominant inheritance.
Fine mapping of Sm
A GWAS analysis was first performed and an interval covering Sm on Chromosome 11 was identified. A total of 30 CAPS and KASP markers were then developed from this 1.7 Mb interval for fine mapping of the Sm gene (Table S2). A total of 1385 F2 individuals were used screen recombinants. Two flanking markers (M29 and M41), whose genetic distances from the Sm gene, were 0.6 and 0.3 cM, respectively, were used to identify recombinants (Fig. 2A-B). Based on the analysis of the F2:3 phenotype of 10 recombinants, as shown in Fig. 2, the Sm gene was finally mapped between marker M390 and marker M410, and the physical distance was about 160 kb (Fig. 2C-D). As shown in Table 1, a total of 10 genes were predicted in the candidate region (Fig. 2E), according to the tomato genome annotation in SGN website (Table1). However, no nonsynonymous SNPs between the two parental lines was found in the CDS regions of the seven genes (Solyc11g020000, Solyc11g020003, Solyc11g020007, Solyc11g020030, Solyc11g020040, Solyc11g020050, Solyc11g020060). Further, no dramatic expression difference of these seven genes was detected between the two parent lines. These results reduced the possibility that the seven genes may be the candidates for Sm. Interestingly, we found that three clustered NBS-LRR resistance genes Solyc11g020080 (named as R1), Solyc11g020090 (named as R2) and Solyc11g020100 (named as R3) in this interval. The amino acid lengths of R1 (126 aa) and R2 (73 aa) proteins are much shorter than R3 (819 aa) (Fig. 2F)(Fig. S2A), suggesting that R1 and R2 might be pseudogenes. This notion was further supported by the gene expression analysis, as the expression levels of these two genes were much lower than R3. More importantly, we didn’t identify any nonsynonymous SNPs between the parent lines in R1 and R2, excluding the possibility that they may be Sm.
Table1 Annotated genes in the Sm region
No.
|
Gene
|
Physical position
|
Functional notes
|
1
|
Solyc11g020000
|
10016462…10017506
|
LOW QUALITY:Acyl-CoA N-acyltransferases superfamily protein, putative isoform 2 (AHRD V3.3 *-* A0A061G7B6_THECC)
|
2
|
Solyc11g020003
|
10024031…10024797
|
Core-2/I-branching beta-1,6-N-acetylglucosaminyltransferase family protein (AHRD V3.3 --* AT1G62305.1)
|
3
|
Solyc11g020007
|
10024816…10027512
|
LOW QUALITY:Serine/threonine protein phosphatase 7 long form isogeny (AHRD V3.3 *** A0A151TRB0_CAJCA)
|
4
|
Solyc11g020030
|
10028013…10028392
|
Lysine--tRNA ligase (AHRD V3.3 *-* A0A0V0IK21_SOLCH)
|
5
|
Solyc11g020040
|
10036469…10040967
|
heat shock protein 70
|
6
|
Solyc11g020050
|
10041183…10041617
|
LOW QUALITY:Cytosolic Fe-S cluster assembly factor nar-1 (AHRD V3.3 -** A0A1D1Z971_9ARAE)
|
7
|
Solyc11g020060
|
10051045…10058502
|
D-3-phosphoglycerate dehydrogenase (AHRD V3.3 *** A0A0B2QT46_GLYSO)
|
8
|
Solyc11g020080 (R1)
|
10154490…10155240
|
Disease resistance protein (AHRD V3.3 *-* A0A103Y7R3_CYNCS)
|
9
|
Solyc11g020090 (R2)
|
10155241…10155462
|
Disease resistance protein (AHRD V3.3 *-* A0A103XRK3_CYNCS)
|
10
|
Solyc11g0200100 (R3)
|
10156748…10161584
|
Disease resistance protein (AHRD V3.3 *** A0A118JXS4_CYNCS)
|
R3 is a candidate gene for Sm
We therefore paid more attention on R3 to verify the candidate gene of Sm. We first noticed that the expression of R3 was significantly induced in response to S. lycopersici inoculation, indicating that this NBS-LRR gene may be involved in the defense response to S. lycopersici. Besides, R3 showed higher expression levels in the resistant line Motelle than that in the susceptible parent line Moneymaker at 48hpi (Fig. 3A). In particular, we found three nonsynonymous SNPs in the CDS region of R3, which caused 3 amino acid changes (S394R, E722K and L782I) from Mm to M (Fig. 3B). While the S394R mutation was located at the WHD domain within the NB-ARC region (also known as NBD-HD1-WHD domain), the other two amino acid changes were located at the LRR domain. To investigate the possible roles of these three amino acid mutations, we carefully compared the protein sequences of the resistant (Mt) and susceptible (Mm) versions of R3 with other NBS-LRR proteins in tomato and Arabidopsis. This analysis indicated that the AA394 may be a critical site that affect the function of R3 and other NBS-LRR proteins. We noticed that, while the Mt version of R3 and other NBS-LRR proteins have conserved positively charged amino acids (Arginine R or Lysine K ) at AA394, the Mm version of R3 at this site is serine (S), a non-charged amino acid. This analysis suggest that the K/R at AA 394 might be very critical for NBS-LRR proteins. These results also led us to a hypothesis that the NBS-LRR protein R3 is non-functional in the cultivated tomatoes due to a critical amino acid mutation, which might be a consequence of selection during tomato domestication. This hypothesis is consistent with the fact that the Sm-conferred resistance can be inherited in a dominant manner.
Before further verifying whether R3 is Sm, we analyzed its cellular sub-localization. The A. tumefaciens GV3101 strain containing the pCAM35::R3-GFP fusion construct was used to infect N. benthamiana leaves. The results showed that R3 was localized to the cytoplasm, nucleus, and cell membrane (Fig. 3C), consistent with previous reports about the cellular sub-localization of NBS-LRRs.
Functional analysis of R3
To further verify R3 as the underlying gene of Sm to confer high resistance to S. lycopersici, we thought to downregulate the expression of R3 in the resistant line Mt to see whether the resistance is abolished or not. VIGS experiments were performed for this purpose. All three clustered NBS-LRR genes R1, R2 and R3 were amplified to construct specific TRV::R1, TRV::R2 and TRV::R3 VIGS vectors. Three weeks after agroinfiltration, the resistant line Mt plants exhibited photobleaching symptoms, demonstrating the silencing was successful. To test whether the gene silencing is specific, expression levels of R1, R2 and R3 in TRV0 (the vector control), TRV::R1, TRV::R2 and TRV::R3 plants were analyzed. As shown in the Fig. 4A, this analysis demonstrated the gene silencing is specific and the silenced plants can be used in further pathogen infection assays.
The TRV::R1, TRV::R2, TRV::R3, and the empty control vector (TRV::00) were inoculated with S. lycopersici. As shown in Fig. 4B, no obvious susceptible symptoms were observed on TRV::R1, TRV::R2 and the empty control vector (TRV::00) plants at 3 days post-inoculation (dpi). By contrast, severe disease lesions were observed in TRV::R3 plants at 3 dpi. Commonly, hypersensitive cell death plays a central role in plant innate immune responses with pathogen restriction. HR (Hypersensitivity reaction) cell death and accumulation of H2O2 were investigated using trypan blue and DAB(3,3ʹ-diaminobenzidine) staining, respectively. Fig. 4C shows that the HR was impaired and hyphal numbers were enhanced in TRV::R3 plants compared with TRV::00 plants at 24 hpi, and the effects were more obvious at 48 hpi. In addition, the accumulation of H2O2 was lower in the TRV::R3 plants than in the TRV::00 plants at 24 hpi. Together, these results provide compelling evidence that R3, a NBS-LRR resistance protein, is involved in Sm-mediated resistance and is the underlying gene of Sm for conferring high resistance to S. lycopersici.
The applicability of CAPS marker in MAS breeding
The CAPS marker M38 (the position is basically the same as M390), which is closely linked with the Sm gene, was chosen to test whether it can be used for accurately distinguishing resistant and susceptible tomato germplasm materials. As shown by agarose gel electrophoresis in Fig. 5, while homozygous resistant plants had two DNA bands (399 bp and 101 bp) and the homozygous susceptible plants has only one DNA band with 500 bp, the heterozygous plants showed three DNA bands with 399 bp, 101 bp, and 500 bp, respectively. Overall, the CAPS marker M38 showed an accuracy rate of 98.96% when used for evaluating 96 tomato accessions (including resistant and susceptible cultivars). These results indicated that this marker can accurately distinguish resistant and susceptible tomato cultivars and can be used as a molecular marker for MAS breeding.