Analysis Of Maize Chlorotic Mottle Virus Pathogenicity And Exploring The Mechanism Of Synergistic Infection With Sugarcane Mosaic Virus

Maize lethal necrosis disease(MLND)is one of the most important viral diseases of maize.MLND occurs when Maize chlorotic mottle virus(MCMV)coinfects the same plant with Sugarcane mosaic virus(SCMV)or several other potyviruses.Recently,the disease has re-emerged and is rapidly spreading to several other areas over the world and threatening the maize production.This reemergence has raised questions about the unknown molecular mechanisms of MLND.In this study,proteomics and viral quasispecies analysis were performed to investigate the molecular mechanism of MCMV and SCMV synergism.Firstly,isobaric tags for relative and absolute quantitation(iTRAQ)assay was conducted to analyze maize(B73)response to MCMV infection and synergistic infection with SCMV at early phase of MLND.Several target proteins that showed differential expression in protein level were selected,and virus induced gene silencing(VIGS)in maize was used to explore their roles in MCMV infection and synergistic infection with SCMV.Totally 605 proteins were identified to be differentially expressed after maize infection with MCMV,among them 407 were up-regulated and 198 were down-regulated.Transcriptional level analysis was carried out by real-time RCR,and we found 16 out of 20,the alteration of protein level was consistent with transcriptional level in response to MCMV infection.KEGG pathway annotation and enrichment analysis revealed ribosome-and photosynthesis-related proteins were significantly enriched.The functions of PDIL1-1 PRX5 and SUS1 in MCMV infection were further investigated by CMV-VIGS system.Their expression levels were reduced to 54%,45%and 56%respectively compared with the control groups after application of virus induced gene silencing.MCMV was then mechanically inoculated onto maize plants,and virus accumulation and symptom development were monitored by time.Silencing of PDIL1-1 and PRX5 significantly inhibited MCMV genomic RNA and virion accumulation accompanied with attenuated symptoms,while there was no significant influence on MCMV infection after silencing of SUS1.Compared with single MCMV infection,723 differentially expressed proteins were identified in synergistic infection with SCMV.Differentially expressed proteins were clustered into 57 GO items by GO ontology annotation and enrichment analysis.Furthermore,KEGG pathway annotation and enrichment analysis of differentially expressed proteins in the synergistic infection arrived 5 highly enriched pathways.Moreover,in the process of MLND formation TCA cycle,y-aminobutyric acid metabolism and hormone-related pathways were significantly altered.ZmPHT3;3/4 and ZmPAP are important proteins in phosphates(Pi)assimilation and in our research,we found both of them were up-regulated in synergistic infection.Reduction in accumulation levels of MCMV and SCMV were found on ZmPHT3;3/4 silencing plants.However,elevations in accumulation levels of both viruses were found on ZmPAP silencing maize plant.We suspect intracellular Pi homeostasis was destroyed after silencing of ZmPHT3;3/4,and also resulted in induced expression of ZmPAP and thus acc-umulation of MCMV and SCMV was inhibited.By now,little information is available on the effects of virus genome variation and evolution in the synergistic infection.In this research,we performed RNA-seq technology to investigate MCMV and SCMV quasispecies in synergistic infection.Furthermore,we also detect alterations of viral quasispecies after serial passages(9 passages)on B73 by mechanical imoclulation.After SNVs(Single Nucleotide Variants)calling,we found viral variation level was slightly higher in noncoding regions along with MCMV genome in the first generation(Ml),and SNVs tended to evenly distribute along coding and noncoding regions after serial passages to the ninth generation(M9).Next,the variation levels of MCMV and SCMV in synergistic infection were also analyzed.We found that the variation level of MCMV was decreased in synergistic infection which can be interpreted as effective enrichment of mutants that were suitable in synergistic infection.Further studies showed an increase in the ratio of transversions in synergistic infection and growing trendency in the course of serial passage.The most frequent transition in MCMV and SCMV was A?G(Adenine?Guanine).And there was more apparent tendentiousness in transversions to T?G(Thymine?Guanine).The ratios of transitions to transversions were 3.3 and 3.9 in the first(S1)and ninth generation(S9)of SCMV single infection respectively.SNVs with higher variable frequency(>1%)were selected to further analysis.Six SNV hotspots were detected in the MCMV genome from M1,M9,MS1 and MS9,indicating they are hotspots of spontaneous mutations in MCMV genome.Further analysis found that three SNV hotspots in MCMV genome in the first generation(M1)and all of them were changed both in synergistic infection with SCMV and in the process of serial passages.After passaged to the ninth generation by mechanically inoculation,ten SNV hotspots were found.And among them the first five were located in P32 ORF resulting in the alteration of four amino acids in P32.When mixture of MCMV and SCMV were passaged to the ninth generation(MS9)by mechanical transmission,seven SNV hotspots were found.It's worth noting that the three SNVs in P32 ORF were all lead to alterion in amino acids.Fairly large variability of P32 ORF was observed whether in MCMV single or in synergistic infection with SCMV,indicating P32 was under higher selection pressure in our experimental conditions.In addition,CP was the most conserved protein in MCMV since no SNVs in CP ORF could introduce alteration in amino acids;while 3' UTR was the region with the highest variation level in MCMV.Compared with MCMV,the 3' UTR of SCMV tended to gain less variation in the synergistic infection with MCMV.In contrast with MCMV,the CP protein of SCMV was under selective pressure in serial passages.Several amino acids in CP of SCMV were changed in the process of serial passages.In the first generation of SCMV single infection there was the only one SNV hotspot,while in the ninth generation five SNV hotspots located were found,and all these six SNVs mentioned above induced alteration in amino acids.We also found two SNV hotspots and one SNV hotspot of SCMV genome in the first and ninth generation synergistic infection with MCMV respectively.These results indicate that the CP of SCMV tends to accumulate more aberrance in the process of single SCMV passage.Interestingly,the variation level in SCMV CP ORF was suppressed in synergistic infection with MCMV,indicating the selective pressure and variation may correlate with the mode of transmission in nature.Viruses encode one or more RNA silencing suppressors to suppress plant RNA silencing-mediated defense.In this study,we found P7a and CP encoded by MCMV were two weak suppressors with the functions of inhibiting local gene silencing using Agrobacterium tumefaciens mediated GFP transient expression system.Furthermore,we showed P7a and CP could suppress systemic gene silencing via inhibition of the transmission of silencing signals.Bimolecular fluorescence complementation(BiFC)assay found P7a and CP can interact with SGS3(Suppressor of Gene Silencing 3),respectively.The finding that the locations of P7a and CP were changed in the presence of SGS3,indicating that P7a and CP may suppress gene silencing by compromising the function of SGS3 through protein interaction.