Host RNAi-mediated silencing of Fusarium oxysporum f. sp. lycopersici specific-fasciclin-like protein genes provides improved resistance to Fusarium wilt in Solanum lycopersicum.

Tomato transgenics expressing dsRNA against FoFLPs act as biofungicides and result in enhanced disease resistance upon Fol infection, by downregulating the endogenous gene expression levels of FoFLPs within Fol. Fusarium oxysporum f. sp. lycopersici (Fol) hijacks plant immunity by colonizing within the host and further instigating secondary infection causing vascular wilt disease in tomato that leads to significant yield loss. Here, RNA interference (RNAi) technology was used to determine its potential in enduring resistance against Fusarium wilt in tomato. To gain resistance against Fol infection, host-induced gene silencing (HIGS) of Fol-specific genes encoding for fasciclin-like proteins (FoFLPs) was done by generating tomato transgenics harbouring FoFLP1, FoFLP4 and FoFLP5 RNAi constructs confirmed by southern hybridizations. These tomato transgenics were screened for stable siRNA production in T0 and T1 lines using northern hybridizations. This confirmed stable dsRNAhp expression in tomato transgenics and suggested durable trait heritability in the subsequent progenies. FoFLP-specific siRNAs producing T1 tomato progenies were further selected to ascertain its disease resistance ability using seedling infection assays. We observed a significant reduction in FoFLP1, FoFLP4 and FoFLP5 transcript levels in Fol, upon infecting their respective RNAi tomato transgenic lines. Moreover, tomato transgenic lines, expressing intended siRNA molecules in the T1 generation, exhibit delayed disease onset with improved resistance. Furthermore, reduced fungal colonization was observed in the roots of Fol-infected T1 tomato progenies, without altering the plant photosynthetic efficiency of transgenic plants. These results substantiate the cross-kingdom dsRNA or siRNA delivery from transgenic tomato to Fol, leading to enhanced resistance against Fusarium wilt disease. The results also demonstrated that HIGS is a successful approach in rendering resistance to Fol infection in tomato plants.

Chauhan S ，Rajam MV 《-》

RNAi-mediated down-regulation of fasciclin-like proteins (FoFLPs) in Fusarium oxysporum f. sp. lycopersici results in reduced pathogenicity and virulence.

Fusarium oxysporum f. sp. lycopersici (Fol) is majorly responsible for causing vascular wilt disease in tomato by blocking transpirational pull, thereby interfering and suppressing overall host immune response. This is mainly achieved by fungal invasion and colonization within the host, resulting in hyphal formation that instigates secondary infection response inside the plant. Earlier reports show the role of fasciclin-like proteins (FLPs) in cell-to-cell adhesions and signaling cascade. Moreover, deletion mutant of FLPs explained its role in development stages of Magnaporthe oryzae and Lentula edodes. Therefore, in present study, based on bioinformatic analysis, we have identified putative FoFLP genes encoding Fusarium-specific fasciclin like proteins. We have exploited the RNAi technology to analyse and understand role of these FoFLPs in virulence during wilt disease in tomato. Interestingly, the morphogenesis of fungal RNAi transformants of FoFLP1, FoFLP3, FoFLP4 and FoFLP5 showed significant reduction in spore count and spore germination frequency, thereby suggesting role of FoFLPs in conidiation. Furthermore, we have detected FoFLP1, FoFLP3, FoFLP4 and FoFLP5 specific siRNAs in the respective fungal transformants, using stem-loop RT-PCR and northern hybridization suggesting targeting of cognate FoFLPs transcripts. Moreover, the fruit invasion and plant infection assays using FoFLP fungal transformants showed late onset of disease with significant reduction in disease symptoms in the infected plants. Although the FoFLP-RNAi fungal transformants entered the host plant by penetrating root cortex, the infected plants showed minimum fungal colonization as a result of siRNA-mediated targeting of endogenously expressed FoFLPs. This further inhibited the propagation of fungal RNAi transformants within root cortex and affected its ability to cause secondary infection response, which led to reduced disease incidence and disease severity index. Altogether, our results show that FoFLPs might play an important role in conidiation and pathogenicity, and can serve as potent RNAi targets. Therefore, Host-Induced Gene Silencing (HIGS) can effectively be used to target FoFLPs in order to raise tomato transgenics resistant to Fusarium wilt.

Chauhan S ，Rajam MV 《-》

Identification of I-7 expands the repertoire of genes for resistance to Fusarium wilt in tomato to three resistance gene classes.

The tomato I-3 and I-7 genes confer resistance to Fusarium oxysporum f. sp. lycopersici (Fol) race 3 and were introgressed into the cultivated tomato, Solanum lycopersicum, from the wild relative Solanum pennellii. I-3 has been identified previously on chromosome 7 and encodes an S-receptor-like kinase, but little is known about I-7. Molecular markers have been developed for the marker-assisted breeding of I-3, but none are available for I-7. We used an RNA-seq and single nucleotide polymorphism (SNP) analysis approach to map I-7 to a small introgression of S. pennellii DNA (c. 210 kb) on chromosome 8, and identified I-7 as a gene encoding a leucine-rich repeat receptor-like protein (LRR-RLP), thereby expanding the repertoire of resistance protein classes conferring resistance to Fol. Using an eds1 mutant of tomato, we showed that I-7, like many other LRR-RLPs conferring pathogen resistance in tomato, is EDS1 (Enhanced Disease Susceptibility 1) dependent. Using transgenic tomato plants carrying only the I-7 gene for Fol resistance, we found that I-7 also confers resistance to Fol races 1 and 2. Given that Fol race 1 carries Avr1, resistance to Fol race 1 indicates that I-7-mediated resistance, unlike I-2- or I-3-mediated resistance, is not suppressed by Avr1. This suggests that Avr1 is not a general suppressor of Fol resistance in tomato, leading us to hypothesize that Avr1 may be acting against an EDS1-independent pathway for resistance activation. The identification of I-7 has allowed us to develop molecular markers for marker-assisted breeding of both genes currently known to confer Fol race 3 resistance (I-3 and I-7). Given that I-7-mediated resistance is not suppressed by Avr1, I-7 may be a useful addition to I-3 in the tomato breeder's toolbox.

Gonzalez-Cendales Y ，Catanzariti AM ，Baker B ，Mcgrath DJ ，Jones DA ... -  《-》

Fol-milR1, a pathogenicity factor of Fusarium oxysporum, confers tomato wilt disease resistance by impairing host immune responses.

Although it is well known that miRNAs play crucial roles in multiple biological processes, there is currently no evidence indicating that milRNAs from Fusarium oxysporum f. sp. lycopersici (Fol) interfere with tomato resistance during infection. Here, using sRNA-seq, we demonstrate that Fol-milR1, a trans-kingdom small RNA, is exported into tomato cells after infection. The knockout strain ∆Fol-milR1 displays attenuated pathogenicity to the susceptible tomato cultivar 'Moneymaker'. On the other hand, Fol-milR1 overexpression strains exhibit enhanced virulence against the resistant cultivar 'Motelle'. Several tomato mRNAs are predicted targets of Fol-milR1. Among these genes, Solyc06g007430 (encoding the CBL-interacting protein kinase, SlyFRG4) is regulated at the posttranscriptional level by Fol-milR1. Furthermore, SlyFRG4 loss-of-function alleles created using CRISPR/Cas9 in tomato ('Motelle') exhibit enhanced disease susceptibility to Fol, further supporting the idea that SlyFRG4 is essential for tomato wilt disease resistance. Notably, our results using immunoprecipitation with specific antiserum suggest that Fol-milR1 interferes with the host immunity machinery by binding to tomato ARGONAUTE 4a (SlyAGO4a). Furthermore, virus-induced gene silenced (VIGS) knock-down SlyAGO4a plants exhibit reduced susceptibility to Fol. Together, our findings support a model in which Fol-milR1 is an sRNA fungal effector that suppresses host immunity by silencing a disease resistance gene, thus providing a novel virulence strategy to achieve infection.

Ji HM ，Mao HY ，Li SJ ，Feng T ，Zhang ZY ，Cheng L ，Luo SJ ，Borkovich KA ，Ouyang SQ ... -  《-》

The tomato I-3 gene: a novel gene for resistance to Fusarium wilt disease.

Plant resistance proteins provide race-specific immunity through the recognition of pathogen effectors. The resistance genes I, I-2 and I-3 have been incorporated into cultivated tomato (Solanum lycopersicum) from wild tomato species to confer resistance against Fusarium oxysporum f. sp. lycopersici (Fol) races 1, 2 and 3, respectively. Although the Fol effectors corresponding to these resistance genes have all been identified, only the I-2 resistance gene has been isolated from tomato. To isolate the I-3 resistance gene, we employed a map-based cloning approach and used transgenic complementation to test candidate genes for resistance to Fol race 3. Here, we describe the fine mapping and sequencing of genes at the I-3 locus, which revealed a family of S-receptor-like kinase (SRLK) genes. Transgenic tomato lines were generated with three of these SRLK genes and one was found to confer Avr3-dependent resistance to Fol race 3, confirming it to be I-3. The finding that I-3 encodes an SRLK reveals a new pathway for Fol resistance and a new class of resistance genes, of which Pi-d2 from rice is also a member. The identification of I-3 also allows the investigation of the complex effector-resistance protein interaction involving Avr1-mediated suppression of I-2- and I-3-dependent resistance in tomato.

Catanzariti AM ，Lim GTT ，Jones DA 《-》