Study On The Synergism Between Southern Rice Black-streaked Dwarf Virus And Rice Ragged Stunt Virus

Southern rice black-streaked dwarf virus(SRBSDV) and Rice ragged stunt virus(RRSV) are members of Reoviridae; SRBSDV is a tentative species in the genus Fijivirus, transmitted by the white-backed plant hopper(WBPH, Sogatella furcifera,),RRSV belong to Oryzavirus vectored by the brown plant hopper(BPH, Nilaparvata lugens).Both two vectors transmit virus in a persistent, circulative-propagative manner. SRBSDV was first discovered in Yangxi County, Guangdong Province, China, in 2001, and identified in 2008. Over the past few years, SRBSDV has rapidly spread throughout southern China and northern Vietnam, and has become one of the most important rice pathogens in these regions. RRSV was first described in China in 1978, it has been distributed sporadically,with severe occurrences in some years and localized outbreaks in others. With SRBSDV’s spread, however, RRSV has become increasingly common in China, and is frequently found in co-infection with SRBSDV. The co-infection may lead synergism between two viruses. Viral synergisms lead to aggravated symptoms by increased the number of cells infected by the virus or the virus titers in the individual cells, and cause significant damage to the agricultural production, besides, viral synergisms also may lead epidemic of new disease, in some cases, it may lead host to loss of the resistant ability of virus. This study was conducted to uncover how SRBSDV and RRSV interacted when co-infection. The results obtained are as follows:1. SRBSDV and RRSV interact synergistically, not only lead aggravated symptoms,but also raised the replication quantity of both virus in co-infected rice plants. By observing symptoms of rice plants, show that rice plants co-infected with both viruses displayed lower plant heights and earlier symptoms. 13 genes of SRBSDV and 11 genes of RRSV were detected using RT-q PCR at 9, 15 and 20 dpi after rice plants singly and mixed inoculation with SRBSDV and RRSV. It is indicated that the expression levels of SRBSDV P5-1、P7-1、P9-1、P10 and RRSV P3、P6、 P8、P10 were increased significantly in co-infected rice plants, those up-regulated genes included some genes associated with forming the matrix of viral factory(viroplasma) of SRBSDV and RRSV. Hinted that, synergismbetween SRBSDV and RRSV increased the replication of the two viruses.2. Co-infection of SRBSDV and RRSV increased virus acquisition efficiency of both vectors. Compared with SRBSDV and RRSV single infection, virus acquisition rates of WBPH and BPH fed on co-infected rice plants at 9,15 and 20 dpi increased 7.4%、27.8%、21.2% and 11.5% 、 19.3% 、 15.3% respectively. Indicated that synergistic interaction between SRBSDV and RRSV increased the virus acquisition efficiency of their respective vectors, and would most likely promote the SRBDV outbreaks and RRSV epidemics.3. Synergism between SRBSDV and RRSV led to rice cells express more serious cytopathologic changes. By using ultrathin section observation of electron microscopy, we found that whether in early, middle or later of the disease progression, the cytopathologic of rice plants co-infection with SRBSDV and RRSV expressed more significant changes than rice plant within singly infection of SRBSDV or RRSV, including that the number of both virus particles and viroplasm increased significant in phloem cells from tip of the first flat leaf of diseased rice plant; chloroplast were markedly swollen; and co-infection also significantly increased the amount of mitochondrias and osmiophilic granule; lipid droplet size increased much more obvious than singly infection. Indicated that synergism between SRBSDV and RRSV need more energy, it may affect host’s lipid metabolism, and lead greater damage4. Compare with SRBSDV or RRSV infection, co-infection of the two viruses alters the expression patterns of key genes in rice RNAi pathway. Using q RT-PCR, we analyzed eight DCLs, nineteen AGOs and five RDRs expression level in the virus infected rice plants.The results showed that co-infection of the two virus could increase the induction rate of Os AGO11 and Os AGO18. Co-infection will have significantly higher expression level of Os AGO11 than SRBSDV infected alone, on 9, 15 and 20 dpi, will have 1.26 fold, 1.55 fold and 1.83 fold of expression level, respectively; but it is not significantly higher than RRSV infected alone. Infection alone of the two virus will not induce the expression of the eight Os DCLs, but three of them(Os DCL2 a, Os DCL3 b and Os DCL4) can be significantly induced by co-infection. Both the two virus infected alone can induce the expression of Os RDR1 and Os RDR4, and infected together can significantly increase the induction. On 9,15 and 20 dpi, the expression level of Os RDR1 in co-infected rice plants are 7.81 fold, 9.00 fold and 9.98 fold higher than SRBSDV infected alone, 8.35 fold, 9.69 fold and 10.14 fold higher than RRSV infected alone, respectively; The expression level of Os RDR4 in co-infected rice plants are 4.56 fold, 9.11 fold and 10.76 fold higher than SRBSDV infectedalone, 4.82 fold, 5.56 fold and 5.83 fold higher than RRSV infected alone, respectively.These results indicated that the synergism between SRBSDV and RRSV deeply influences the RNAi pathway of the rice plants.5. The two RNA silencing suppressors of each virus have the additive effect. By coinfiltration of agrobacterium on transgenic Nicotiana benthamiana plants 16 C, we analyzed the GFP silencing suppressing activity of SP6 from SRBSDV and RP6 from RRSV working alone or together. One day post infiltration, only 19.83% and 30.42% area of SP6 or RP6 infiltrated alone shows green fluorescence, respectively, and 89.02% area of SP6 and RP6 infiltrated together shows green fluorescence. Meanwhile, the m RNA level of GFP in the co-infiltration area significantly higher than SP6 or RP6 single infiltration area, rising to 3.36 fold and 3.18 fold, respectively. Two days post infiltration, the green fluorescence signal reached the peak in all treatment, infiltration alone or together of SP6 and RP6 show no significantly difference of m RNA level of GFP. Three days post infiltration, the green fluorescence signal become weaker in SP6 or RP6 single infiltration area, and the co-infiltration area keeps strong green fluorescence signal. Meanwhile, the m RNA level of GFP in the co-infiltration area significantly higher than SP6 or RP6 single infiltration area,rising to 2.04 fold and 1.39 fold, respectively. These results shows that working together of the two RNA silencing suppressors could significantly advance the suppressing of RNA silencing, increase the effect of suppressing and extend the period of suppressing, it might be the mechanism of the synergism between the two viruses.The above findings, clearly explain the high incidence of SRBSDV and RRSV co-infection in field, suggest the possibilities that the two viruses could mutually improve the epidemic spread of each other, and provide new clues for the investigation of the molecular mechanism of virus synergism.