Essential Roles Of The Myosin V In Plant Cotton-verticillum Interaction

Object: Cotton(Gossypium hirsutum)is the most important economic crops in world and the main source of natural fibers.However,Verticillium Wilt is the major disease of cotton,which seriously affects the yield and quality of cotton and restricts the production of cotton.Verticillium wilt is a soil-borne vascular fungal disease,which is one of the most devastating threats to more than two hundred of dicotyledonous plants,including the economic crops of cotton,tomato,lettuce etc.Cotton Verticillium wilt,which is mainly caused by Verticillium dahliae,has become a major obstacle to cotton production in China.V.dahliae is a soil-borne,semi-biotroph filamentous fungus.Given that V.dahliae colonizes in the host plant xylem system and its the resting form,microsclerotia,can live in soil for more than ten years,there still lack effective measures to control the Verticillium wilt.Thus far,numerous pathogenicity-related genes in V.dahliae have been identified by genetic,molecular and biochemical methods.In particular,studies on the infection structure have achieved a significant progress.V.dahliae is predicted to secret seven hundreds of secretory proteins,but few of them was validated to be an effector or a virulence factor,which is tightly associated with the pathogenicity.However,the secretion process in Verticillium is remains not unclear.While the secreted proteins may have diverse functions,resulting in the complexity of the relative studies,the secretion components associated with the secretory pathway are relatively conserved.Thus,in the present study,we will mainly explore the roles of the secretory components of Verticillium during plant infection.The exocytosis is a conventional secretory pathway of eukaryotic cells based on vesicle transport,the motor protein Myosin V provides power and energy to vesicles transport along the actin.The Myosin V-mediated motility system has been well studied in yeast and Aspergillus nidulans,but keeps unexplored in Verticillium during plant infection.Here,we investigated the roles of Myosin,a V.dahliae ortholog of Myosin V,in the process of pathogenicity.Method:(1)According to the published sequence of the Myosin V protein in model filamentous fungi Aspergillus nidulans,we found the V.dahliae Myosin2(Myo2),an ortholog of Myosin V.Then,we constructed the phylogenetic tree of MyoV in filamentous fungi.(2)Using the gene knockout techniques based on the homologous recombination and Agrobacterium tumefaciens-mediated transformation,we generated the knockout mutant strain ?Vdmyo2,and investigated the colony morphology,germination rate and mycelial growth mode.(3)The spatial and temporal dynamics of Myo2,which was further validated by the complement test,were observed by spinning disc confocal microscope.(4)The invasion processes of V.dahliae were uncovered by scanning electron microscopy.(5)The pathogenicity test of the ?Vdmyo2 mutant strain was analyzed by the injection method in cotton and by the dipping root method in Arabidopsis thaliana,respectively.We compared the pathogenicity of mutant and control wild type strains.(6)The secreted proteins of V.dahliae(in both wild type strain and the ?Vdmyo2 mutant strain)were identified by iTARQ proteome analyses and mass spectrometry.The differential proteins were screened by the Scaffold 4.0 software.Function annotation,GO analysis and functional classification of the differential proteins were analyzed by the Blast2 GO softwareResults and conclusions:(1)The Myo2 shows a high homology with other members of the Myosin V family in filamentous fungi.?Vdmyo2 knockout mutant strains show severe growth defects,with the significantly reduced of white mycelium,increased melanin and microsclerotia,and delayed of growth.In addition,conidial morphology and mycelial were seriously affected.Conidia are round;the hyphae lost the polarity,and display threedimensional growth,and thicker,hyperbranch and hyperseptation.These results show that Myo2 involved in conidia development and growth polarity of hyphae.(2)Myo2-GFP is located in the cytoplasm with a dot-like structure and in the apex of the hyphae with a round-shaped structure,which colocalizes with the Spitzenk?rper marker FM4-64,further indicating that Myo2 plays an important role in the vesicle transport process.(3)The pathogenicity of the ?Vdmyo2 mutant strain was found to be significantly reduced during infecting host cotton and Arabidopsis thaliana.The ?Vdmyo2 mutant strain cannot colonize in the vascular system of cotton stem,and viable fungi were not isolated from stem sections.This result indicates that Myo2 is required for the pathogenicity of V.dahliae.In addition,we found that the mutant strain could still invade the Arabidopsis cells.We speculated that the reduced pathogenicity may not be caused by the impairment of the invasion process,but be related to the defective secretion.(4)We further compared the cotton-induced secretome between the ?Vdmyo2 and the wildtype strain through iTRAQ and mass spectrometry,the results showed that the expression of secretory proteins,which are involved in carbohydrate transport and modification and intracellular transport,secretion,vesicle transport is down-regulated due to Myo2 mutation.Taken together,these results uncovered that Myo2 mainly regulates the secretory pathway to influences pathogenicity of V.dahliae,thus plays a key role in plant-Verticillium interaction.