| Maize chlorotic mottle virus(MCMV)could cause a severe disease,corn lethal necrosis disease(CLND)when synergistically interacted with Maize dwarf mosaic virus(MDMV),Wheat streak mosaic virus(WSMV)or Sugarcane mosaic virus(SCMV).In this study,we found that CLND in China was caused by co-infection of MCMV and SCMV,and MCMV or CLND distributed only in Yunnan and Sichuanprovinces in China.With purified MCMV virus particles as immunogen,monoclonal antibodies and polyclonal antibodies against MCMV were raised,and based on the antibodies,three high efficient detection methods for MCMV,including TAS-ELISA,DIBA and IC-RT-PCR,were established.Five full-length cDNA of MCMV found in Yunnan and Sichuan provinces were cloned.Aphylogenic tree was constructed with all the available full-length cDNA sequences.All the Chinese isolates shared 96.9-97.3%identity to the American isolates,and the Chinese isolates were clustered to one group whereas the two American isolates formed another group.Identity analysis using all the available CP sequences showed that,Chinese isolates shared over 99%identity with Thailand isolates and East African isolates.We constructed an infectious cDNA clone of MCMV using the full-length cDNA of the MCMV YN2 isolate.The cDNA of YN2 was inserted into the binary vector pCB301 and the construct was termed pMCMV.Following agroinfitration,the infectious clone pMCMV highly efficently infected maize plants.The symptoms induced by pMCMV were indistinguishable to that of the wild type virus,and the relative virus accumulation in the plants inoculated with pMCMV was comparable to that of wild type virus.Sperical viral particles resembled those in wild type virus infected plants were observed in the crude extracted sap of pMCMV infected plants under a transmission electron microscope,and the recombinant MCMV was sap transmissible to healthy maize seedlings by rub-inoculation.We compared the transcriptome and cytopathology differences between MCMV single-infection and MCMV and SCMV co-infection.On the transcriptome level,more differentially expressed genes(DEGs)were detected in MCMV and SCMV co-infected plants as compared with those from MCMV infected plants,MCMV and SCMV co-infection induced 7,247 DEGs while MCMV induced 2,916 DEGs.Among these DEGs we focused on DEGs related to plant defence reactions.For example,many genes in the pathway of oxylinpins(such as Jasmonic Acids)synthesis,salicylic acid(SA)synthesis and auxin synthesis were expressed differentially,and these DEGs may be responsible,at least in part,for the different symptoms between MCMV infection and MCMV and SCMV co-infection.Cytopathology differences were analyzed with transmission electron microscopy(TEM),MCMV and SCMV co-infection induced more severe changes on cellular level.Much smaller starch grains in chloroplasts of bundle sheath cells were observed,and mitochondrias were destroyed earlier in CLND cells as compared to MCMV cells.TEM showed that chloroplast photosynthesis was impaired and mitochondrial respiration was disrupted more quickly in CLND plants than MCMV plants.The more severe cytopathology changes might shed light on the mechanism of CLND caused by MCMV and SCMV co-infection.Multivesicular structures resembled peroxisomal multivesicular bodies(pMVBs)induced by Tomato bushy stunt virus(TBSV)were observed in both MCMV infected and MCMV and SCMV co-infected plants.In addition,both p50 and p111 of MCMV,which were similar to the replication-related protein p33 and p92 of TBSV respectively,located on the peroxisome membranes when fused to green fluorescence protein(GFP),p50 interacted with p111 directly in a yeast two-hybrid assay.The results indicated that pMVBs induced by MCMV might be the replication sites of MCMV,and p50 and p111 were both involved in the process of replication machinary assembling.Similar as the p33 gene of TBSV,p50 was predicted to contain two transmembrane domains(TMs),which might be important for its localization to peroxisomes. |
