The Mechanism Of Tumorigenesis In Plants Infected By Southern Rice Black-streaked Dwarf Virus And The Role Of Tumors In The Viral Infection Cycle

Southern rice black-streaked dwarf virus(SRBSDV)or Rice black-streaked dwarf virus 2(RBSDV-2)is a novel virus classified in the genus Fijivirus,family Reoviridae.In recent years,the virus has been epidemic and has caused serious yield losses of rice and maize in Vietnam and China.Typical symptoms on rice and maize plants include pronounced stunting,darkening of leaves,and white waxy or black-streaked swellings or tumors along the veins of leaf or stems.Like SRBSDV,most plant reoviruses,including fijiviruses,oryzaviruses,and phytoreoviruses,induce the development of swelling,galls,enations or tumors in the veins of host plants.However,the mechanisms of symptom development,especially the mechanism of tumorigenesis,have remained unknown until now.In this study,the tumors induced by SRBSDV in rice plants were used as a model and a series of microscopical,molecular biological,and reverse genetic methods were employed to analyze the structure and biogenesis of virus-induced tumors in plants.Histochemical staining and microscopical studies showed that SRBSDV-induced tumors were derived from hyperplasia of the phloem.The hyperplastic region could be divided into two parts,cytoplasm-rich and-absent parts.Quantitative assays showed that the tumors contained higher levels of viral RNAs and proteins,indicating that tumors are places that contribute to viral multiplication and accumulation.In the tumors,a number of viroplasms,virus arrays,and tubular structures were found,supporting the above hypothesis and indicating that the tumors could play an important role in the infection cycle of SRBSDV.Comparisons of the ultrastructure of SRBSDV-induced tumors with that of phloem in healthy plants illustrated how the normal development pattern was obviously modified in tumors.In the tumors,the cytoplasm-rich region was mainly composed of phloem parenchyma(PP)and the cytoplasm-absent region was composed of sieve elements(SEs)without companion cells(CCs),whereas the classical staggered pattern of SE and CCs was always observed in the phloem of healthy plants,indicating that the cellular arrangement of tumors does not comply with the traditional SE-CC pattern.In morphology,the SEs of healthy phloem were always oriented in a consistent direction and they had a high ratio of long axis to short axis,usually about 30.In contrast,SEs in tumors elongated in different directions and the ratio of long axis to short axis was usually less than 3,indicating that the development of SEs in tumors was abnormal.A number of viral proteins,nucleic acids,and particles were detected in SEs and PPs of tumors,suggesting that both SEs and PPs in tumors could sustain viral multiplication and accumulation.Thus these modified SEs provide SRBSDV a large space to multiply,indicating that the formation of tumors is helpful for the viral infection cycle.In order to investigate the role of tumors in the viral infection cycle and the movement of the viruses with large-sized virions,such as SRBSDV(80 nm in diameter),we at first focused on the gateways between cells in tumors.Significantly,a novel kind of gateway was found on the cellular interfaces within the tumors.The new gateways were abundant on the interfaces of SE-SE and SE-PP in the tumor but only a few were found in healthy plants.Structurally,the new cellular gateways are more similar to the plasmodesmata(PD)than to the sieve pores.The gateway has a central pith module,which resembles the desmotubule(DT)of PDand is also composed of actin,but,like the sieve pore gateway,it allows passage of the SRBSDV virion,which is 80 nm in diameter and much larger than the size exclusion limit of normal PD(about 30 nm).Gold labelling assays showed that the gateway could modify the adjacent cell wall components,decreasing the amount of cellulose and increasing the amount of callose.In this study,the novel gateway was tentatively designated as the flexible(FL)gateway in view of its adjustable channel property.In tumors,the frequency of FL gateways significantly increased and their distribution was much more dispersed,potentially enhancing symplastic transport among SEs and PPs.Observation by electron microscopy revealed that the large SRBSDV virion cannot be involved in PD,but they were observed within the FL gateways on the SE-SE or SE-PP interfaces,providing direct evidence that the FL could be involved in viral movement.Ultra-structures of plants infected by other plant reoviruses were further investigated.We found that all tested phloem-limited reoviruses,namely Rice black-streaked dwarf virus(RBSDV),Rice gall dwarf virus(RGDV),and Rice ragged stunt virus(RRSV),caused a similar hyperplasia of phloem,within which the cellular types and organization and the frequency of FL gateways were all similar to those of SRBSDV.By contrast,the ultra-structure of plants infected with RDV,a non-phloem-limited phytoreovirus,was similar to that of healthy plants.Based on these results,we proposed a working model for the roles of tumors in the infection cycles of phloem-limited plant reoviruses and the means by which FL gateways facilitate the movement of plant reoviruses in plants.To further understand the mechanism of tumorigenesis in virus-infected plants,we constructed cDNA libraries of rice and SRBSDV and then found an interaction using the yeast two hybrid system between SRBSDV P1 and a homolog of retinoblastoma in rice(OsRBR1),a factor of cell cycles.Both bimolecular fluorescent complementation(BiFC)and pull-down assays consistently showed that there was a strong interaction between P1 and OsRBR1 in vivo and in vitro.Assays using truncated mutants showed that the C-terminal part of P1 and the pocket A and B domains of OsRBR1 were crucial for their interaction.Immunogold labeling assays showed that both P1 and OsRBR1 were co-localized within viroplasms,indicating that the interaction could change the subcellular localization and distribution of the protein.Transient expressing of P1 in Nicotiana benthamiana leaves significantly interfered with the interaction between RBR and E2F proteins.Over-expression of P1 in tobacco using the recombinant virus vector and in rice using transgenic method not only affected the expression of genes downstream of the RBR-E2F signal pathway,but also induced hyperplasia of cells in the vascular bundles,producing the swellings or tumors on the veins of leaves.However,plants over-expressing a mutant P1 that could not interact with OsRBR1 appeared to be similar to the control plants.In addition,P1 overexpression in tobacco also induced an increase of the frequency of SE related intercellular gateways(including PPU and flexible gateway)as comparing to the negative control.The P1 mutant without the capability of interacting to RBR leaded to a recovery of frequency of SE related intercellular gateway,which resembled to the negative control.Over-expressing P1 in rice plant by a transgenic system also induce cellular hyperplasia in phloem.These results indicate that SRBSDV P1 is a viral oncogene in plants,which interferes with the subcellular localization and distribution,RBR-E2F interaction,and expression of downstream genes by interacting with RBR,and thus changes the cell cycles and induces tumors.This study has therefore revealed the structural features of tumors,discovered a novel intercellular gateway that is involved in the cell-to-cell movement of plant reoviruses,identified the oncogene of SRBSDV in plants and the mechanism by which SRBSDV P1 induces cell hyperplasia by its interaction with plant RBR.These results would contribute to a deeper insight into viral movement,the mechanism of tumor-induction in plants and the RBR-E2F pathway itself.