Characterizing The Function Of V2 And C4 Encoded By TYLCV In Virus Movement And Pathogenicity

Tomato yellow leaf curl virus(TYLCV),as a monopartite begomovirus of the family Geminiviridae,is transmitted by whiteflies and causes one of the most devastating plant diseases worldwide.Since first report in Shanghai in 2006,this virus has spread to many tomato production regions of China and became one of the most serious threats on local tomato production.TYLCV contains a single genome component with six open reading frames(ORFs).C1(Rep),C2(TrAP),C3(REn)and C4 are located on the complementary strand,and the other two ORFs(V1 and V2)are located on the viral strand.Three viral proteins,including V1,V2,and C4,are possibly associated with virus movement.V1 functions as a nuclear shuttle protein to help virus movement between the nucleus and the cytoplasm,C4 is proposed to facilitate viral cell-cell movement with unclear host factors and unknown mechanisms,V2 also has a cryptic role in virus movements.As V2 also acts as one of the most important pathogenicity determinants with unknown pathways,consequently,we choose V2 and C4 as the main research object,trying to clarify the roles in virus movement,the mechanism involved in the pathogenicity induced by V2 and the mechanism of C4 protein involved in virus movement during virus infection.In order to analyse the function of the TYLCV V2 protein,V2 fused with YFP(V2-YFP and YFP-V2)were constructed and infiltrated into plant cells via agrobacterium-mediated transient transformation.The subcellular localization of V2 in plant cells was observed via confocal microscopy and the results showed that the V2 protein mainly localizes in the cytoplasm and perinuclear areas.The formation of aggregates/inclusion bodies was also found in cytoplasm,which promoted that different aggregate forms may affect the function of the V2 protein.Besides,the V2 protein was found co-localized with ER,actin and microtubules,which are well known as playing important roles in protein transportation,suggesting that the V2 protein might be involved in the virus intra-and inter-cellular trafficking during viral infections.Except for cytoplasmic distribution,YFP signal was also found in perinuclear areas and in the nucleus when we checked the subcellular localization of V2,while the signal in the nucleus was extraordinary weak.Since the V2 protein contained a nuclear export signal in its N terminal,the NES was important for perinuclear localization of V2,suggesting that V2 might act as a nuclear shuttle protein to help virus traffic from the nucleus to the cytoplasm.V1 protein encode by TYLCV is well known as a viral genomic DNA binding protein and functions as a nuclear shuttle protein.To clarify the role of V2 protein in the normal function of V1,the direct and indirect interaction between V1 and V2 was tested and the results revealed that V2 could bind V1 directly.When co-expressing V1 and V2 in N.benthamian cells,V2 can promote the expression of V1 protein and change the subcellular localization of the V1 protein,causing less nuclear distribution,which suggested that V2 protein might be involved in the VI-mediated export of viral genomic DNA from the nucleus,through direct binding with VI or V1-ssDNA complex.Further,we also found the self-interaction of V2 when testing the interaction between V2 and other proteins encoded by TYLCV,which was reinforced by Y2H,BiFC and Co-IP.To identify the key motif of V2 involved in its self-interaction,the results confirmed that the S71A mutation of V2 abolished its self-interaction,which indicates the serine on position 71 of V2 is essential for the self-interaction.Besides,research on the subcellular localization of V2S71A revealed it also shared cytoplasmic and perinuclear distributions,which was similar with wide type V2,however,the number of large cytoplasmic aggregates decreased.To clarify the possible pathway involved in the decreased number of aggregates,oryzalin,latrunculin B and MG 132 were introduced.When treated with MG 132,the number of cytoplasmic aggregates increased and showed similar levels compared to wild-type V2.The results indicated that mutation S71A on V2 might affect 26S proteasome-mediated degradation of the large cytoplasmic aggregates,proposing that the self-interaction of V2 might be involved in a counter-defence response for TYLCV to protect V2 aggregates against host pant 26S proteasome-mediated degradation.As V2 was involved in the virus symptom modulation,the effect of self-interaction on the pathogenicity of V2 was also tested.In PVX heterogenous system,V2 could induce severe symptoms increasing the accumulation of PVX,while V2S71A only caused mild mosaic symptoms,which was similar to the symptoms induced by the empty PVX vector.The effects of S71A to the pathogenicity of TYLCV was further investigated by infectious clones and the mutation S71A on V2 was found associated with lower viral DNA accumulation and mild symptom.These results indicated that the serine on position 71 is essential for the pathogenicity of V2,which suggests there might be a correlation between the self-interaction and the pathogenicity of the V2 protein.Besides as viral factors,V2 was also reported interaction with some host factors to fulfil its function,such as SGS3 and CYP1.A new host factor,s1GRXC6,was found based on the results of yeast two-hybrid screening using V2 as a bait.The full length of s1GRXC6 was cloned via RT-PCR using total RNA isolated from TYLCV-infected tomato as a template,which encoded a CC type glutaredoxin with a conserved CCMC motif.The interaction between V2 and s1GRXC6 and the full length this host factor were further confirmed by Y2H,BiFC and Co-IP,which showed V2 could interact with s1GRXC6 in vitro and in vivo.Truncated mutants of V2 were constructedbased on the functional domain of the V2 protein,and results showed that C85A mutation of V2 abolished its interaction with s1GRXC6,suggesting the cysteine on position 85 of V2 is the key site for the interaction.What's more,C85A mutation on V2 affected the perinuclear localization,caused few large cytoplasmic aggregates and weakened the pathogenicity,suggesting that the interaction between V2 and s1GRXC6 might be involved in the pathogenicity of the V2 protein.As V2 interacted with s1GRXC6,to characterize the effects of the interaction on their subcellular localization,V2 fused with RFP and s1GRXC6 fused with YFP were introduced.s1GRXC6 was localized in the cytoplasm and nucleus while V2 was localized in the cytoplasmic and perinuclear regions.Co-infiltrating with s1GRXC6,V2 was also found in the nucleus,which showed that the interaction between V2 and s1GRXC6 changed the subcellular distribution of V2,indicating that the interaction might occur in both the cytoplasm and the nucleus.The conclusion is further confirmed by the result of BiFC experiments.In order to investigate the biological function of the interaction between V2 and s1GRXC6,the expression pattern of s1GRXC6 during the TYLCV infection or the single V2 expression was examined and the results showed that the expression of V2 promoted the expression of s1GRXC6.However,overexpression of s1GRXC6 induced host plants becoming higher and slowing down the disease symptoms development and viral genome DNA accumulation when inoculated by TYLCV.Silencing S1GRXC6 induced host plants showing dwarf symptom,promoted virus symptoms development and virus genome DNA accumulation when inoculated with TYLCV.It seems that the expression of V2 can activate the expression of tomato s1GRXC6 gene and host defence-response to protect plants form virus attacks.However,overexpression of V2 using PVX heterogenous system or transgenic assay could not only induce upregulation of s1GRXC6,but also dwarf and yellow symptoms.Further research on the accumulation of s1GRXC6 proteins revealed that most of s1GRXC6 proteins were bound by V2 and present in the form of s1GRXC6-V2 complex,which caused the low level of original free s1GRXC6 proteins and subsequently induced the development of dwarf symptom in host plants.In order to investigate the function of s1GRXC6,yeast two-hybrid assay was introduced to screen possible candidates interacting with s1GRXC6.A tomato thioredoxin reductase,NTRC80,was found interacting with s1GRXC6.To characterize the effects of V2-s1GRXC6 interaction and s1GRXC6-NTRC80 interaction,MBP-tagged NTRC80,GST-tagged s1GRXC6 and His-tagged V2 were expressed and purified respectively in vitro,the results showed NTRC80 pulled down by s1GRXC6 was reduced with increasing amounts of V2 protein.These results provide evidences that V2 could bind s1GRXC6 in a competitive manner with NTRC80,which indicated that V2 could interact with s1GRXC6 and interfere the recruitment of NTRC80 by s1GRXC6,subsequently affecting host plant growths,developments and developing dwarf symptoms.In the end,since C4 protein was reported as a movement protein in TYLCV with an unclear mechanism,we constructed YFP-tagged C4 to further investigate the function of it in virus movement.The results showed that the signal mainly localized in the cell cytoplasm and plasma membrane and co-localized with plasmodesmata.To characterize the host candidates involved in C4-mdeiated virus movement,cDNA library screening was introduced using C4 as the bait and a new host factors,s1COP,was found.s1COP is a tomato coatmer,which could form membranous related complex,playing an important role in protein transport process.The interaction of C4 and s1COP was further confirmed by yeast two-hybrid assay and BiFC.s1COP was mainly located in nuclear membrane and cytoplasm.TYLCV infection or C4 expression upregulated the expression of s1COP and over-expression of s1COP enhanced the virus genomic DNA accumulation and accelerated the development of disease symptom during TYLCV infection,indicating that C4 could promote the expression of s1COP and help virus movement in host plants.All these results prompt us to an idea that C4 might utilize s1COP-mediated vesicular transport systems through direct interaction with s1COP to fulfil its function in cell-cell movement of TYLCV.