| Cucumber(Cucumis sativus)is one of the most important facilities vegetable crops in China.With high yield,rich nutrition and high economic value,cucumber is playing more and more important role in enriching the vegetable basket of residents,meeting the annual supply of vegetables,getting rich in rural areas and increasing farmers' income.However,in recent years,with the adjustment of cultivation patterns and the change of environment,the virus diseases on cucumber have appeared frequently,which has caused serious problems for the safety production of cucumber crops.Cucurbit chlorotic yellow virus(CCYV)is a kind of plant virus transmitted by whitefly,which infects many kinds of cucurbit crops.Since the virus was introduced into China in 2010,it has caused serious losses to the production of melon,watermelon,cucumber and other crops in many provinces and cities.In 2016,when we investigated the virus diseases on cucumbers in Shouguang and other places in Shandong Province,we found that there was a disease on the local cucumbers with yellow and slight dwarf.In order to identify the accompanying virus types,we carried out molecular detection on the disease samples collected in the field,and detected CCYV from it.Further CP gene cloning and sequence analysis also confirmed this result.This is the first report of the virus infecting cucumber in Shandong Province.On the one hand,Shandong Province is an important vegetable production base in China.The occurrence of CCYV may cause serious threat to the local cucumber production.On the other hand,Shandong Province is also an important seedling base in China.The nationwide seedling transportation is likely to further aggravate the spread of CCYV in a wider range,so the damage of this disease on cucumber deserves close attention.In order to further clarify the characteristics and taxonomic status of the isolate and lay a foundation for further study on the pathogenic mechanism of the virus.In this study,the genome of CCYV was cloned by segmentation method,and the sequence information of RNA1 and RNA2 of CCYV Shandong isolate was obtained by splicing after sequencing.The results of sequence analysis that RNA1 was 8,607 bp in length,RNA2 was 8,041 bp in length,encoding 12 proteins in total;sequence homology analysis showed that RNA1 was highly homologous with other CCYV isolates reported,and the homology was more than 99.5%;cluster analysis also showed that they were clustered into CCYV branches,which indicated that the virus isolated from cucumber in Shandong Province was CCYV.This is the first report of the genome sequence of CCYV on cucumber crops in Shandong Province.Many research results show that the subcellular location of the virus-encoded protein is the basis and place for its function,so studying subcellular location of virusencoded protein is beneficial to promote the analysis of virus-encoded protein function.However,there are few reports on the cell localization of CCYV encoded proteins.In order to determine the subcellular distribution of CCYV encoded proteins,we cloned 9 proteins and constructed the expression vector with YFP fluorescent label.After infiltrating Nicotiana benthamiana leaves,we observed them with laser confocal microscope.The results showed thatshowed that P6-1,P4.9,P9,CP,and CPm could be transcribed and expressed normally in cells(Western blot)among 9 proteins.These proteins were distributed in cytoplasm and nucleus,and CPm was enriched in nucleolus,while the remaining P22,P26(weak expression),P6-2,HSP70 h and other genes were not well expressed(not detected by Western blot),and their location has not been studied.At present,only P4.9 has been reported in CCYV coding protein.These work laid a foundation for further study of its role in the process of virus infection and pathogenicity.Although CCYV encodes many proteins,there are few functional reports.In order to study the role of CCYV encode proteins in the process of virus pathogenesis,10 genes(RNA1 encodes P6-1 and P22,RNA2 encodes(CP,CPm,HSP70 h,P59,P26,P9,P6-2,P4.9)were cloned in this study,and the pathogenic characteristics of potato X virus(PVX)heterologous viral vectors were studied,The results showed that 8 genes could cause more serious symptoms on the infiltrated and inoculated N.benthamiana : p6-1 induced obvious yellow,shrinkage and necrosis of new leaf;P22 induced systemic necrosis;p4.9 induced shrinkage and necrosis of upper leaf;P6-2 induced shrinkage and necrosis of upper leaf;P59 induced shrinkage and necrosis of new leaf;CP induced system leaves appear shrinkage and necrosis spots;CPm induced severe chlorosis and yellow in the upper leaves;P26 induced chlorosis and mosaic in the upper leaves of the plants;while PVX inoculated control group showed only slight mosaic symptoms,suggesting that these genes may be involved in the process of virus infection and pathogenicity.However,the other two genes(HSP70h,P9)caused similar symptoms on N.benthamiana to the control group,which may not be directly involved in symptom formation as pathogenic factors.In order to further analyze the mode of action of the candidate pathogenic factors encoded by CCYV,this study use the yeast two-hybrid system to study the interaction between the 8 candidate factors obtained in the previous screening and other proteins encoded by the virus.The results showed that among the 110 combinations screened,P22,P59 and P6-2 interact with each other,and the three of them all caused necrosis when heterologous overexpression occurs.The interaction of the three may be beneficial to mutual regulation and and inhibition of their necrosis reaction in virus infection.At the same time,interaction between P6-2 and P59 and HSP70 h is also found,suggesting that HSP70 h may be involved in inhibiting the necrosis reaction;P59 itself has interaction,It may be that the polyproteins are involved in the interaction between virus the host.To sum up: CCYV was isolated from cucumber in Shandong Province,and used as a research object to clone and sequence the whole genome to clarify its genomic structure characteristics,Meanwhile,the subcellular localization characteristics of its coding protein were studied by confocal observation,and the pathogenic characteristics of its coding protein were studied by heterovirus expression system,It was clarified that the eight encoded proteins would aggravate the symptoms of the disease,and it is a candidate factor related to pathogenicity.And the yeast two-hybrid technique was used to screen the virus-encoded proteins that interacted with it,and the possible mode of action in the process of virus infection was preliminarily discussed,which laid the foundation for the in-depth analysis of the mechanism of the CCYV-encoded protein in the pathogenesis process.The foundation also provides a reference for the subsequent targeted development of virus prevention and control strategies that target the key processes of viral pathogenicity. |
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