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 《-》

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 《-》

Fusarium oxysporum f. sp. lycopersici C(2)H(2) transcription factor FolCzf1 is required for conidiation, fusaric acid production, and early host infection.

Yun Y ，Zhou X ，Yang S ，Wen Y ，You H ，Zheng Y ，Norvienyeku J ，Shim WB ，Wang Z ... -  《-》

Host-Induced Silencing of Pathogenicity Genes Enhances Resistance to Fusarium oxysporum Wilt in Tomato.

Bharti P ，Jyoti P ，Kapoor P ，Sharma V ，Shanmugam V ，Yadav SK ... -  《-》

Identification of hub genes and potential networks by centrality network analysis of PCR amplified Fusarium oxysporum f. sp. lycopersici EF1α gene.

Fusarium wilt is a devastating soil-borne fungal disease of tomato across the world. Conventional method of disease prevention including usage of common pesticides and methods like soil solarisation are usually ineffective in the treatment of this disease. Therefore, there is an urgent need to identify virulence related genes in the pathogen which can be targeted for fungicide development. Pathogenicity testing and phylogenetic classification of the pathogen used in this study confirmed it as Fusarium oxysporum f. sp. lycopersici (Fol) strain. A recent discovery indicates that EF1α, a protein with conserved structural similarity across several fungal genera, has a role in the pathogenicity of Magnaporthe oryzae, the rice blast fungus. Therefore, in this study we have done structural and functional classification of EF1α to understand its role in pathogenicity of Fol. The protein model of Fol EF1α was created using the template crystal structure of the yeast elongation factor complex EEF1A:EEF1BA which showed maximum similarity with the target protein. Using the STRING online database, the interactive information among the hub genes of EF1α was identified and the protein-protein interaction network was recognized using the Cytoscape software. On combining the results of functional analysis, MCODE, CytoNCA and CytoHubba 4 hub genes including Fol EF1α were selected for further investigation. The three interactors of Fol EF1α showed maximum similarity with homologous proteins found in Neurospora crassa complexed with the known fungicide, cycloheximide. Through the sequence similarity and PDB database analysis, homologs of Fol EF1α were found: EEF1A:EEF1BA in complex with GDPNP in yeast and EF1α in complex with GDP in Sulfolobus solfataricus. The STITCH database analysis suggested that EF1α and its other interacting partners interact with guanosine diphosphate (GDPNP) and guanosine triphosphate (GTP). Our study offers a framework for recognition of several hub genes network in Fusarium wilt that can be used as novel targets for fungicide development. The involvement of EF1α in nucleocytoplasmic transport pathway suggests that it plays role in GTP binding and thus apart from its use as a biomarker, it may be further exploited as an effective target for fungicide development. Since, the three other proteins that were found to be tightly associated Fol EF1α have shown maximum similarity with homologous proteins of Neurospora crassa that form complex with fungicide- Cycloheximide. Therefore, we suggest that cycloheximide can also be used against Fusarium wilt disease in tomato. The active site cavity of Fol EF1α can also be determined for computational screening of fungicides using the homologous proteins observed in yeast and Sulfolobus solfataricus. On this basis, we also suggest that the other closely associated genes that have been identified through STITCH analysis, they can also be targeted for fungicide development.

Tripathi YN ，Singh VK ，Kumar S ，Shukla V ，Yadav M ，Upadhyay RS ... -  《BMC MICROBIOLOGY》