Insight Into The Colonization Process And Exoproteome Of Verticillium Dahliae Infection On Monocots And Eudicots

Verticillium dahliae is a typical soil-borne vascular bundle pathogenic fungus.It has a wide host range,can invade more than 600 dicotyledons,including more than 180 important agricultural crops.However,it can only harm dicotyledons but not monocotyledons.Part of previous studies have shown that V.dahliae can't recognize and germinate on the surface of monocotyledons maize and rice roots after one week of inoculation.However,it is still unclear whether V.dahliae can infect monocotyledons and the process of infection.At the same time,it is unclear the properties of secreted proteins during interaction with monocotyledons.This study identified pathogenicity of V.dahliae on monocotyledon maize,wheat,sorghum and dicotyledons cotton,tomato,tobacco.The results further confirmed that V.dahliae can harm dicotyledons,causing the typical symptoms of Verticillium wilt.The dicotyledons root system was underdeveloped,vascular bundle was brown;the growth rate of V.dahliae at the junction of cotton stem and rhizome was significantly increased and a large amount of V.dahliae biomass was isolated from cotton plant stem.However,V.dahliae could not cause the typical symptoms of Verticillium wilt and had almost no effect on the growth and development of monocotyledons maize,wheat and sorghum.Scanning electron microscopy(SEM) was used to observe the germination and expansion of V.dahliae on the root tip surface of cotton and maize.In the early stage of infection(6 h-24 h),the conidia germination on the root tip surface of maize and cotton was same,but in the middle and late stage of infection(48 h-120 h),the conidia germination rate on cotton was significantly faster than that of maize with more hyphae.Transmission electron microscopy(TEM)was used to observe the infection dynamics of V.dahliae inside the root tips of cotton and maize.It was found that there was no significant difference in the infection process between cotton and maize at the early stage(epidermis and cortical stage,24 h to 48 h).However,the cell walls of the mycelia and conidia inside the maize root tips began to crumple and degrade at the late stage of infection(the middle column vessel stage,5 d to 10 d),and the cells contents began to lose.After 10 d of infection,a large number of mycelia and conidia died due to the loss of the contents.These indicated that V.dahliae could invade into the maize roots,but couldn't survive and reproduce therein.SEM was further used to observe the ability of upward expansion of V.dahliae on the roots of cotton and maize.It was found that V.dahliae could colonize and germinate on the root surface,extend from the root cake joint to the base of the hypocotyl and invade from the surface to the interior of both cotton and maize seedling roots.However,the mycelium was significantly limited in maize roots than that cotton.Fungal biomass quantification also confirmed the significant difference between two plants.Above results showed that compared with dicotyledon cotton,V.dahliae can infect monocotyledon maize root system and extend from the surface into internal region of root tip,but in the late infection V.dahliae conidia and hyphae cannot survive for a long time and V.dahliae lose ability if infection.The purpose of this study was to clarify the differences of the extracellular proteins of V.dahliae during infecting cotton and maize.337 proteins were found expressed differentially under the cotton and maize roots induction condition by extracellular proteome analysis and comparation of V.dahliae.Compared to water control,200 proteins were specifically expressed under cotton roots inducting condition,65 proteins were specifically expressed under maize roots inducting condition,and 72 proteins were differentially expressed under both cotton and maize roots inducting conditions.Furthermore,comparing with extracellular proteins induced by maize,V.dahliae secreted more extracellular proteins related to cell wall degradation,oxidative stress,inhibition of host immune response,and pathogenicity under the cotton induction conditions indicated by GO,KEGG,Interpro and Cazymes database annotations.In order to clarify the contribution of differentially expressed secreted proteins of extracellular proteome during V.dahliae infecting cotton and maize,these genes function was analysed.Among the40 differentially secreted proteins specifically induced by cotton,abundance of five proteins were significantly increased.After the overexpression of the five secreted protein genes with significantly increased abundance,the overexpressed transforming strains were inoculated into maize roots and their infection ability to maize root tips was observed by transmission electron microscopy.The results showed that the overexpression of Vd Csp1(peptidase M28),Vd Csp3(peptidase M14)and Vd Csp4(oxidoreductase)significantly enhanced the infection ability of V.dahliae to maize.The conidia of V.dahliae in maize root tips had stronger persistent infection ability,and the degree of necrosis of the conidia was significantly reduced.Moreover,the secreted protein Vd Csp2 with significantly increased abundance inhibited the function of PAMPs Vd EG1-induced tobacco necrosis phenotypes.Among the19 differentially secreted proteins specifically induced by maize,abundance of one proteins was significantly increased.After the gene(Vd Msp1)was genetically knocked out which was significantly increased abundance.The ability of infecting maize root tips was significantly reduced and the conidia of V.dahliae in maize root tips were more quickly necrotic and disinfected.At the same time,it was found that secreted proteins Vd Msp9,Vd Msp11 and Vd Msp13 with abundance variation more than 5folds specifically induced by maize can manipulate host immunity.Among the 30 differentially secreted proteins co-induced by cotton and maize,abundance of protein Vd CMcp1(unknown function)was significantly increased under both conditions.The virulence of the knockout mutant against cotton was significantly decreased after inoculation of cotton.In conclusion,V.dahliae can infect and harm the dicotyledonous cotton,but it only infect the monotyledonous maize with no typical Verticillium wilt symptoms.During V.dahliae infecting to monocotyledons and dicotyledons,it can break through the structural barriers and manipulate the immune response of host/non-host plants by secreting different functional effector proteins to realize the invasion to non-host plants and the harm to host plants.