Identification Of The Infection Structure Of Phytopathogenic Fungus Causing Eggplant Wilt Disease And The Mechanism Of Cotton MiR171 Confers Resistance To Verticillium Dahliae

Verticillium wilt is a worldwide plant vascular disease caused by soil-borne fungi that leads to yield reduction and even total crop failure in a variety of crops.Verticillium dahliae and Verticillium albo-atrum are the main pathogenic fungi of Verticillium wilt.Dissecting the detailed infection process of pathogenic fungi and the resistance mechanism of plants would facilitate the development of effective strategies for crop protection.In the first part of this study,we identified that V.dahliae is the pathogenic fungus of eggplant(Solanum melongena L.)Verticillium wilt(named as Vsm).Furthermore,we studied the colonization process of Vsm.In the second part of this study,we found that cotton miR171 a can confer resistance to V.dahliae(V592).The colonization process of eggplant roots by Vsm: We isolated the fungal hyphae from the infected eggplants collected from disease field.Internal Transcribed Spacer(ITS)sequencing indicated that the pathogenic fungus is V.dahliae(Vsm).We studied the colonization process of eggplant roots by green fluorescent protein-tagged Vsm.At the early stage,we observed that Vsm spores randomly attach to the root surface and germinate to produce mycelium.The hyphae wrap the root surface,and only a few germinating hyphae penetrate intercellularly into the epidermal cells.During perforation,we observed a slight swelling of the hyphae.Upon successful penetration into the epidermal cells,the hyphae rapidly proliferate and quickly progress up the xylem vessels to the above-ground,developed typical verticillium wilting.To detect whether the slight swelling of the hyphae is a special infection structure,we first incubated Vsm on a cellulose film laid on medium.We observed that Vsm can penetrate the cellophane film and grow on medium.Under microscopy,we found that Vsm develops hyphopodia and penetration pegs to breach the membrane,whereas both the NADPH oxidase gene VsmNoxB and tetraspanin VsmPls1 mutants form hyphopodia without penetration pegs.In addition,the mutants,VsmΔNox B and VsmΔPls1,cannot penetrate cellulose film,which corresponds to reduced pathogenicity compared to Vsm.Furthermore,the confocal microscopy results revealed that VsmNoxB and VsmPls1 are co-localized to the endoplasmic reticulum.During the formation of the penetration pegs,VsmNoxB and VsmPls1 specifically accumulated at the base of hyphopodia.VsmNoxB is essential for producing reactive oxygen species(ROS)by VsmNoxB.Moreover,we demonstrated that VsmNoxB/VsmPls1-mediated ROS production activates the VsmPls1 pathway by regulating calcium signaling to form penetration pegs.The molecular mechanism of miR171 conferring cotton resistance to V.dahliae: In general,miRNAs regulate target genes at the posttranscriptional level by degrading m RNA or inhibiting translation in a sequence specific manner.It is well known that miRNAs play very important roles in plant growth and development,as well as responding to biotic or abiotic stresses.In our previous study,small RNA(s RNA)sequencing data showed that Arabidopsis thaliana infected by V.dahliae(V592 strain)significantly repressed the expression of miR171 c.In this study,Northern blot results showed that V592 infection suppresses the expression of cotton miR171 a.In Arabidopsis,miR171 targets GRAS family transcription factors.We detected increased accumulation of two cotton target genes,A12G0855 and D12G0935,homologs of the Arabidopsis GRAS family SCL6 gene,in V592-infected cotton plants compared to mock infection.Tobacco transient expression assays confirmed that miR171 a are able to suppress A12G0855 and D12G0935 expression.Furthermore,both overexpressed miR171 a and silenced A12G0855 and D12G0935 reduce the susceptibility of cotton to V592.In summary,miR171 family miRNAs confer cotton plant resistance to V.dahliae by suppressing GRAS family gene expression.