Functional Characterization Of Small Gtpase ShROP1 And ShROP11, Microfilament Skeleton Polymerization Factor ShARPC3 And ShARPC5 In The Interaction Between Tomato And Oidium Neolycopersici

Tomato powdery mildew,caused by Oidium neolycopersici?On-Lz?,is a devastating disease in tomato production.However,the mechanism of tomato resistance against On-Lz remains largely undefined.In eukaryotic organisms,signal transduction can be regulated by small G proteins,which affect downstream effectors by switching the activation state of binding GTP and the inactivation state of binding GDP,and then regulate various signal transduction and metabolism processes.Among the small G proteins,the highly homologous proteins of Rho family from plants are plant-specific small GTP binding proteins named Rops.The downstream actin-related protein ARP2/3 complex of Rops family is the main regulator of actin microfilament dynamics,which has been linked to multiple cellular processes,including those associated with response to stress.Therefore,it is significant that the plant immune mechanism mediated by ShROPs and ShARPCs is studied,so as to comprehensively understand the immune mechanism of small G protein and microfilament skeleton,and to further elucidate the molecular mechanism of interaction between tomato and On-Lz.In this study,tomato and Arabidopsis were used as the main research objects.A combination of bioinformatics,virus-induced gene silencing?VIGS?,high performance liquid chromatography?HPLC?,tobacco transient overexpression,Arabidopsis mutant complementation,overexpression in Arabidopsis,subcellular localization,qRT-PCR,and yeast mutant complementation approaches were used to study the functions of ShROP1,ShROP11,ShARPC3 and ShARPC5,and ShROPs interaction proteins were screened by yeast two-hybrid.Following the important results were obtained.1.Bioinformatics analysis showed that the amino acid sequences of small G protein ROP1 is highly conserved.ShROP1 was mainly located in cell membrane and cytoplasm.Compared with the compatible interaction between tomato MM?Moneymaker?and On-Lz,the expression of ShROP1 gene was significantly up-regulated in incompatible interaction between tomato LA1777?Solanum habrochaites?and On-Lz.In VIGS trials,silencing of ShROP1 resulted in enhanced susceptibility to the On-Lz.The content of SA?Salicylic acid?in silenced-ShROP1 plants was significantly decreased by HPLC.Histological observation showed that the accumulation of HR and H₂O₂ could reduce in silenced-ShROP1 tomato and Arabidopsis?Arabidopsis thaliana?rop1 mutant plants compared with wild types,respectively.Transient overexpression of ShROP1 in tobacco can rapidly produce necrotic spot.These results indicated that ShROP1 could induce plants to produce HR and H₂O₂ to resist pathogenic fungi invasion,suggesting that ShROP1 might be involved in ETI defense response,and could also regulate plant disease resistance by relying on SA,an endogenous signaling molecule,to activate the expression of downstream disease resistance-related proteins and destroy the cell structure of pathogenic fungi.2.ShROP11 is mainly located in cell membrane and nucleus.The expression of ROP11gene in the incompatible interaction was significantly higher than that in the compatible interaction.Additionally,silencing ShROP11 significantly reduced the resistance of tomato LA1777 against On-Lz.Compared with the control,the silenced-ShROP11 plants produced less HR and H₂O₂ after inoculation.And the content of ABA?Abscisic acid?in silenced-ShROP11 plants was significantly decreased by HPLC.Interestingly,overexpression of ShROP11 gene in Arabidopsis rop11 mutant could restore the disease resistance of Arabidopsis.It is concluded that ShROP11,as a positive regulator,participates in the response of tomato to biotic stress and inhibits the invasion of pathogenic fungi by inducing plants to produce HR and H₂O₂,and prevents invasion by relying on ABA signaling pathway.3.The interactions between ShROP1 and ShProfilin,ShROP1 and ShCHI9 proteins were confirmed by yeast two-hybrid GAL4 system.These results suggest that ShROP1 may participate in plant PTI pathway by interacting with Profilin protein to regulate downstream actin polymerization.ShROP1 could also inhibit the growth of pathogenic fungi by interacting with ShCHI9 protein by destroying the cell wall of pathogens.Additionally,it was confirmed that ShROP11 interacted with ShCHI3 and ShNP24 proteins,respectively.ShROP11 could regulate the production of antimicrobial substances by interacting with ShNP24 protein,thus participating in the PTI pathway of tomato disease resistance,and also prevent fungi infection by interacting with ShCHI3 protein by destroying the cell wall of pathogens.These results provide a structural basis for the immune function of ShROP1 and ShROP11 proteins.4.Herein,the ShARPC3 gene encoding a subunit protein of the Arp2/3 complex was identified and characterized.ShARPC3 encodes a 174-amino-acid protein possessing a conserved P21-Arc domain.Silencing of ShARPC3 resulted in enhanced susceptibility to On-Lz,demonstrating a role for ShARPC3 in defense signaling.Interestingly,a loss of ShARPC3 coincided with enhanced susceptibility to On-Lz,a process that we hypothesize is the result of a block in the activity of SA-mediated defense signaling.Conversely,over-expression of ShARPC3 in Arabidopsis thaliana,followed by inoculation with On-Lz,showed enhanced resistance,including the rapid induction of hypersensitive cell death and the generation of reactive oxygen.Heterologous expression of ShARPC3 in the arc18 mutant of Saccharomyces cerevisiae?i.e.,?arc18?resulted in complementation of stress-induced phenotypes,including high temperature tolerance.Taken together,these data support a role for ShARPC3 in tomato through positive regulation of plant immunity in response to On-Lz pathogenesis.5.Bioinformatics analysis showed that tomato ARP2/3 complex subunit ShARPC5contains a conserved P16-ARC domain and is closely related to Arabidopsis and tobacco.VIGS trials showed that ShARPC5 could induce ROS and HR in tomato to respond to On-Lz infection,and could induce the expression of PR1,a pathogenesis-related protein.On the contrary,over-expression of ShARPC5 in Arabidopsis increased plant resistance against On-Lz.In addition,ShARPC5 is involved in plant responses to abiotic stresses.These results suggest that Arp2/3 complex plays an important role in disease resistance during pathogenic fungi infection.