The Involvement Of C4 Protein Encoded By Malvastrum Yellow Vein Virus In Viral Pathogenesis

Malvastrum yellow vein virus(MaYVV)is a member of begomoviruses in the family Geminiviridae.MaYVV is associated with cognate betasatellite,Malvastrum yellow vein betasatellite(MaYVB)and infects tomato plants and a variety of weeds,posing a potential threat to tomato production.Further research on the origin,genetic evolution and pathogenic mechanism of MaYVV/MaYVB is of great significance to the field control of MaYVV/MaYVB.Existing researches of MaYVV/MaYVB mainly focus on field sample detection and identification,genetic evolution,virus pathogenicity,etc.Researches on the key pathogenic factors of the virus and its involvement in virus pathogenicity remains to be uncovered.In this study,one of the key pathogenic factors of MaYVV/MaYVB,namely C4 protein encoded by MaYVV,was screened by gene overexpression,and related researches on its role in pathogenicity of MaYVV/MaYVB and its effect on host growth and development were carried out.The effect of C4 on host gene expression pattern and the related pathways was comprehensively analyzed by transcriptome sequencing technology.The host factor JAZ1 was identified interacting with C4 protein by Y2H and Bi FC,and C4 gene expression inhibits JA response of host plants.The effects of JA pathway downstream transcription factors on MaYVV/MaYVB infection were analyzed by gene overexpression and silencing.The main findings in this study are as follows:1.Effect of MaYVV-encoded C4 protein on symptoms induction and virus accumulationBased on the overexpression of MaYVV and MaYVB genes using PVX vector,it is found that?C1,V2,C1,C2,and C4 genes expression aggravate PVX-induced symptoms.Among them,C4 gene overexpression causes bumpy leaf and mosaic symptoms at early stage,and then the plants exhibit upward leaf curling and flower deformity.At 15 dpi,the relative expression of PVX in PVX/C4 plants was significantly higher than that in PVX empty vector plants.Analysis of the MaYVV genome sequence revealed that two potential start codons are existed in C4 gene.The two potential start codons of C4 gene were mutated without affecting the C1 ORF in the MaYVV genome and then inerted into the binary vector p LH9000.When associated with MaYVB,the infectious clone harboring the mutation of the first potential start codon induced similar leaf curl and yellow vein symptoms on plants with MaYVV at 10 dpi.The mutation site was recovered and causes mutations in other sites at 35 dpi;however,when two potential start codons were both mutated(MaYVV?C4),the induced symptoms by MaYVV?C4 inassociation with MaYVB were milder than thise in MaYVV/MaYVB at10 dpi.No mutation recovery was detected on MaYVV?C4/MaYVB inoculated plants at 35 dpi.Subsequently,we used MaYVV?C4 for inoculation alone or associated with MaYVB inoculation on N.benthamiana.At 15 dpi,no obvious symptoms of viral disease were found in MaYVV?C4-or MaYVV-inoculated plants.When associated with MaYVB,the leaf curl symptom induced by MaYVV?C4/MaYVB was milder than that induced by MaYVV/MaYVB at 15 dpi.Virus accumulation was significantly reduced in MaYVV?C4-inoculated plants when compared with that in MaYVV-inoculated plants;both virus and betasatellite accumulation in MaYVV?C4/MaYVB were lower than those in MaYVV/MaYVB inoculated plants.2.C4 expression complements the accumulation deficiency of MaYVV?C4 and alters plant cell growthAfter verifying the C4 m RNA expression,C4 transgenic plants were inoculated with MaYVV alone,with the inoculated wild type plants as control.Results showed that no obvious symptoms of viral disease were found in wild type and C4 transgenic plants,virus accumulation in C4 transgenic plants was significantly higher than that in wild type plants.After inoculation with MaYVV/MaYVB,wild type and C4 transgenic plants began to show symptoms of leaf curl and yellow veins at 10 dpi,and the accumulation of MaYVV and MaYVB in transgenic plants was significantly higher than that of wild-type plants.When C4 transgenic plants were inoculated with MaYVV?C4 and wild-type plants were inoculated with MaYVV,the virus accumulation in transgenic plants was significantly higher than that in wild type plants at 10,15,and 20 dpi;When C4 transgenic plants were inoculated with MaYVV?C4/MaYVB and wild type plants were inoculated with MaYVV/MaYVB,the virus and betasatellite accumulation in transgenic plants was significantly higher than that in wild type plants at 15 dpi.The above results show that C4 protein promotes the accumulation of MaYVV and MaYVB in plants.Because the C4 transgenic plants exhibited upward leaf curling and uneven leaf lamina growth,we examined the resin-embedded leaf cross sections and found that the the size and the arrangement of mesophyll cells in C4 transgenic leaves were significantly altered compared with that in the WT leaf cross sections,such as uneven leaf lamina growth,disordered and larger palisade and spongy cells,and some crowds of mesophyll.DAPI staining and scanning electron microscopy analysis revealed that the number of nucleus,epidermal cells and stomata cells per mm~2 in transgenic C4 plants increased significantly,and the surface area of stomata cells did not change significantly;RT-q PCR analysis of the expression of several genes involve in cell division and expansion showed that cyc D3;1,cyc B1;4,cyc A1;1,upa7 and CDKD3 were significantly up-regulated in transgenic plants when compared with those in wild-type plants.By inoculating on GFP trnsgenic line 16c plants,it was found that transient expression of C4+35S:GFP effectively inhibits PTGS and triggered green fluorescence at 3 dpi.In summary,C4 protein promotes the accumulation of MaYVV and MaYVB,disrupt host cell cycle and inhibit PTGS.3.Transcriptome sequencing analysis of host endogenous gene expression upon C4expressionBased on transcriptome sequencing technology,44338 and 43996 genes were obtained from wild-type and C4 transgenic plants,respectively.3727 genes were differentially expressed in transgenic plants,of which 2007 genes were up-regulated and1,720 genes were down-regulated.These differentially expressed genes(DEGs)were enriched to 1,047 GO terms,in which 124,312 and 971 GO terms were involves in cellular component,molecular function and biological process,respectively.These DEGs are mainly participate in cellular processes,metabolic processes,single-organism processes,and binding,catalytic activity,cells,cell part and other processes.KEGG enrichment analysis of DEGs revealed that they are mainly enriched in plant hormone signal transduction,plant-pathogen interaction,secondary metabolite biosynthesis,metabolic pathways,and so on.Ten differentially expressed genes were randomly selected for verification by RT-q PCR and we found that the expression of these genes were consistent with transcriptome sequencing data.Among the top 20 enriched pathways,?-linolenic acid metabolism,carotenoid biosynthesis,diterpenoid biosynthesis,plant hormone signal transduction and plant-pathogen interaction pathways were affected by C4 expression through altering the expression of related genes.4.MaYVV C4 protein interacts with JA pathway inhibitor JAZ1Bi FC and Y2H assay were conducted and the interaction between C4 and JAZ1was sreened and verified.The isolated Nb JAZ1 protein possesses conserved NT,TIFY and Jas domains,shares high similarity to the JAZ1 proteins from the Solanaceae.Subcellular localization analysis showed that C4 protein was localized in the nucleus and plasma membrane,while JAZ1 was mainly distributed in the nucleus,with weak signals in the plasma membrane.Detection of JAZ1 m RNA in different tissues revealed that the expression level of JAZ1 was highest in flowers,followed by leaves,stem and roots.JAZ1 and JA pathway marker genes PDF1.2 were significantly down-regulated in C4 transgenic plants.Inoculation and detection of MaYVV/MaYVB on Me JA-treated plants revealed that,Me JA-treated plants showed a delay and alleviated symptoms induced by MaYVV/MaYVB.The accumulation of MaYVV in Me JA-treated plants was significantly lower than that in control at 7,10 and 12 dpi,the accumulation of MaYVB in Me JA-treated plants was significantly decreased at 10 dpi when compared with that in control.In summary,the results showed that C4 interacts with JAZ1 and inhibits the JA response.Me JA can effectively inhibit MaYVV/MaYVB infection.5.Effects of C4 gene on the expression of JA-responsive transcription factors and their involvement in MaYVV/MaYVB infectionAnalysis of transcriptome data combined with RT-q PCR,we verified the down regulation of JA-responsive transcription factors WRKY8,WRKY41 and MYB44 in C4transgenic plants compared with those in wild type plants.For further confirming their involvement in MaYVV/MaYVB infection,TRV-based vector was used for silencing WRKY8,WRKY41 and MYB44 gene.After confirming the efficient silencing,plants were inoculated with MaYVV/MaYVB.At 20 dpi,MYB44 silenced plants showed more severe leaf curl symptom than that in the control.The accumulation of MaYVV and MaYVB in MYB44 silenced plants was significantly higher than those of the control.The leaf curl symptom induced by MaYVV/MaYVB and the accumulation of MaYVV and MaYVB in WRKY41 silenced plants were similar to that in the control.At 17 dpi,symptoms induced by MaYVV/MaYVB and the accumulation level of MaYVV and MaYVB in WRKY8 silenced plants were more severe than that in the control.Subsequently,the three genes were overexpressed by PVX-based vector.PVX/MYB44inoculation caused necrosis in leaves and stem,PVX/WRKY8 and PVX/WRKY41caused necrosis in leaves.After confirming the overexpression efficacy,we inoculated these plants with MaYVV/MaYVB.At 20 dpi,symptoms induced by MaYVV/MaYVB on WRKY8 and MYB44 overexpreesed plants were alleviated compared to the PVX contol plants,the symptoms on WRKY41 overexpreesed plants were similar to the PVX contol plants.The accumulation of MaYVV and MaYVB in PVX/WRKY8 and PVX/MYB44 plants were significantly lower than that in the control,while the accumulation of MaYVV and MaYVB in PVX/WRKY41 plants were not significantly altered.In summary,the results indicated that C4-induced JA-responsive genes WRKY8and MYB44 have an inhibitory effect on MaYVV/MaYVB infection,and WRKY41 had no significant effect on MaYVV/MaYVB infection.In this study,C4 protein was found involving in leaf curl symptom formation that induced by MaYVV/MaYVB,and promoted the accumulation of MaYVV and MaYVB in the host.As a key pathogenic factor,MaYVV C4 protein disrupts the host cell cycle and inhibits host defense-related PTGS.Transcriptome analysis showed that multiple endogenous genes was affected by C4 gene expression,of which mainly involves in plant defense.C4 interacts with JAZ1,inhibits the JA response and the corresponding responsive transcription factor expression,thereby promoting MaYVV/MaYVB infection.These results reveal the mechanism of C4 in virus pathogenesis and provide a theoretical basis for studying host resistance to MaYVV/MaYVB infection.