Quantification Analysis Of Cucumber Mosaic Virus Genomic RNAs

Cucumber mosaic virus (CMV) is a typical single-stranded, plus sense RNAvirus, and has an extremely large host range and has a worldwide distribution, one ofthe pathogenicity impaired crops. It has three genomic RNAs. So this simple genomicRNA virus is one of model strains to investigate pathogenicity mechanism. This paperstudies synergy between CMV and ZYMV and absolute quantification of genomicRNA in CMV virus.1. Synergy between Cucumber mosaic virus and Zucchini yellow mosaic virusVirus complex infections occur in both plant and animal systems, and doublyinfected organisms commonly display increases in disease symptoms and in theaccumulation of one or both of the viruses. The characterized viral synergy is theinteraction between CMV and ZYMV. This work studies the synergy between CMVand ZYMV by different views. The results show that: these two viruses complexinfection causes more serve symptom than single infection and the disease indexincreases in complex infection. Real-time RT-PCR results: At 35 dpi, the relativeamounts of CMV-Fny ORFs la, 2a, 3a and CP in the complex infection were 1.48,2.24, 2.51, 0.99 folds of those in the single infection on cucumber plants; the relativeamounts of CMV-Fny ORFs la, 2a, 3a and CP in the complex infection were 15.14,4.90, 3.16, 2.57 folds of those in the single infection on bottle gourd plants. Therelative amounts of ZYMV-SD CP ORF in the complex infection were 90％and 22％of those in the single infection, on cucumber and bottle gourd, respectively. This studybases on real-time RT-PCR; we firstly precisely quantify the accumulation of viralgenomic RNA when CMV and ZYMV infected cucumber and bottle gourd plantssingly or doubly, and establish a relative quantification method, 18S rRNA was usedas an internal control.2. Interaction between Cucumber mosaic virus and Tomato mosaic virusThe interference phenomenon among viruses commonly occurred in naturalenvironment. The first invaded virus usually effected the second one on replication,transportation, assemble and so on. The interaction between CMV and ToMV showedthat: the tomato plants, which were pre-treated with CMV-Fny, successfully obtained resistant ability against the hypervirulent virus ToMV-N5. The double infection plantsdid not show the severe symptoms that induced by ToMV-N5 single infection. In thisstudy, the host biological reaction, some physiological index and accumulation ofviruses were examined in virus single infection or double infection plants. Our resultswould give some evidences for complex infection study between CMV and ToMV,and some basic theories for virus interaction were involved in this work.3. The primary study on function of CMV satRNA isolate from tomato plantSatellite RNAs (satRNAs) are molecular parasites that can modulate pathogenesisof helper viruses. In this study, a satRNA (T1sat) strain was cloned by RT-PCR fromCucumber mosaic virus (CMV) infected tomato and the full length of Tlsat was 337nucleotide (Accession Number: DQ785472). Mixture of CMV-Fny and T1satRNAwas co-inoculated on the seedlings of Nicotiana tabacum, Lycopersicon esculentumand Cucurbita pepo, respectively. At 3 days post-inoculation (dpi), RT-PCR analysisresults showed that Tlsat only existed in the systemic leaves of N. tabacum and L.esculentum. The accumulation of T1sat in the inoculated leaves of host plants wasanalyzed by Real-time RT-PCR and results indicated that the level of T1sat C. pepowas far less than that of N. tabacum or L. esculentum. Therefore, non-existence ofT1sat in the seedlings of C. pepo was due to its low accumulation in the inoculatedleaves. Tlsat attenuated symptoms induced by CMV-Fny on N. tabacurn, and theaddition of T1sat to CMV-Fny inoculum aggravated symptom expression on L.esculentum, while T1sat exerted no effect on symptom on C. pepo caused byCMV-Fny. T1sat depressed the level of CMV-Fny 1a, 2a, 2b, 3a and CP in the.tissuesof N. tabacum or L. esculentum, but the effect of T1sat on CMV-Fny genomic RNAsaccumulation on N. tabacum was more severe than that on L. esculentum. T1sat didnot resulted in decrease of viral genomic RNAs on C. pepo. Our results suggested thatsymptom expression induced by CMV-Fny was not related to the level of viralgenomic RNAs in host plants.4. Absolute quantification ananlysis of genomie RNAs in CMV virionsCMV is a tripartite RNA virus and the genomic RNAs are encapsidated in threetypes of different coat proteins. It is reported that the relative ratios among multipartite virus genomic RNAs have a strong effect on the level of virusaccumulation and pathogenicity. This work establishes standard curves by in vitrotranscription with gradient dilution, and gets a tmitary equation of linear relationshipbetween logarithm of RNA concentration and cycle threshold. So the copies ofgenomic RNA in virions can be calculated according to cycle threshold of respondingCMV RNAs. Results: the copy number ratios between CMV-Fny 1, 2, 3 and 4 invirions were determined to be 1.00:(1.17±0.11):(3.58±0.20):(5.81±0.31). To confirmthe quantification results of real-time RT-PCR, RNAs extracted from virions are alsoanalyzed in parallel by Lab-on-a-Chip and Northern blot hybridization assays, andsimilar relative concentrations are obtained. This study bases on real-time RT-PCR;we firstly precisely quantify the relative ratios among CMV-Fny genomic RNAs, andestablish an absolute quantification method by standard curves to supply the referencefor practice and theory.