Charaterization Of Tospoviruses In China And Their Infectivity Analysis On Arabidopsis Thaliana

Tospovirus is a genus infecting plants in Bunyaviridae.The genome of the viruses in this genus is tripartite negative single strand RNA.Over 20 species belong to this genus which can be further divided into American-and Euro/Asian-type groups according to the geographical distribution.Tomato spotted wilt virus(TSWV)is the type species of Tospovirus and was ranked No.2 in the Top ten plant viruses of scientific and agricultural importance.TSWV has a wide host range including more than 1,000 plants in 82 families.It is transmitted by thrips persistently and circularly every year in the world and causes serious economic losses on agricultural,horticultural and ornamental plants.Arabidopsis thaliana is one of the most important model plants to study the interaction between plant and pathogenic microorganisms because of its comprehensive genome information,perfect genetic system and complete mutant library.In this study,we devoted to the identification of Tospovirus species in Yunnan and Hainan,and on this basis,established the infection system of Tospovirus in Arabidopsis,providing a good genetic research system for the study of virus-host interaction.Five tospoviruses were isolated from Yunnan and Hainan,and the viruses infection system of Arabidopsis was established.It was found that different developmental stages of Arabidopsis had important influence on the infection of tospoviruses,and its preliminary mechanism was analyzed.The main research contents include the following three aspects:1.Identification of tospoviruses in Yunnan and the establishment of Arabidopsis infected systemA field investigation and identification of tospoviruses was carried out in Yunnan area.Tospovirus mainly infects tomato,pepper,tobacco,spider orchid and other economic crops or ornamental plants.The incidence rate of tospoviruses disease in local fields plants achieved 90%.Tomato spotted wilt virus(TSWV),Tomato zonate spot virus(TZSV),Impaliens necrotic spot virus(INSV)and Hippeastrum chlorosis ring spot virus(HCRV)were identified by ELISA and RT-PCR.Host inoculation test of TSWV lettuce strain showed that the strain could infect Nicotiana benthamiana,N.tabacum cv.K326,N.rustica,Capsicum annuum,Datura stramonium,Lactuca sativa,Emilia sonchifolia,Chenopodium.quinoa,Petunia hybrida,Solanum lycopersicum and other plants.Through the concentration test of A.thaliana,symptoms development and observation,re-inoculation to N.benthamiana and detecting the viruses by RT-PCR,the system that TSWV,TZSV and INSV infected into Arabidopsis was successfully established.2.Identification,genome sequencing,origin of specific S RNA intergenic region and establishment of Arabidopsis infected system of Capsicum chlorosis virus from HainanIn this study,tospoviruses was also identified in Hainan.Capsicum chlorosis virus(CaCV)was isolated and identified from spider orchid by universal primers and named CaCV-Hainan.On this basis,the full genomic sequence of CaCV-Hainan was further determined.The full genomic RNA S,M and L contained 3629,4859 and 8912 nucleotides(nt),respectively.Further sequence analysis revealed that the S RNA of CaCV-Hainan had a particularly long intergenic region(IGR)of 1348 nt,whereas M RNA had an IGR of 462 nt.Further sequence analysis of different CaCV isolates from the world revealed that the S RNA IGR of CaCV-Hainan originated from CaCV-Qld-3432,an Australian isolate with a particularly long IGR,through deletion of two stretches of 25-and 325-nt in length.Moreover,the S RNA IGR of CaCV-Hainan was inserted with two stretches of 10-and 20-nt sequences of an unknown origin.The S RNA IGR of CaCV-Ph from Taiwan and CaCV-NRA from Thailand also derived from the CaCV-Qld-3432 through deletion of 218-nt sequences.Usually there is a significant IGR variation in the Tospovirus species,but the S RNA IGRs in CaCV species varies greatly.Our findings provide valuable new insight into the structural variations and evolutionary origin of CaCV IGRs.In addition,we established the system that CaCV successfully infected into A.thaliana Col-0 by mechanical inoculation.3.Infection rate of tospoviruses on Arabidopsis thaliana determined by different developmental stages and the preliminary mechanism exploration of this phenomenonMature resistance of plants to pathogenic microorganisms is a very common phenomenon.When plants are young,they are susceptible to be infected by pathogens.When plants grow to maturity,they are susceptible to be infected by pathogens.However,in this study,we found the opposite situation that Arabidopsis was less susceptible to be infected by tospoviruses at young stage.With the development of Arabidopsis,the infection rate of tospoviruses to Arabidopsis increased.When 3-week-old A.thaliana Col-0 was inoculated with TSWV-LE isolate,it usually can not be infected.In 4,5 and 6-week-old A.thaliana Col-0,the infection rates of TSWV-LE isolate were 9%,21%and 25%,respectively.But all plants inoculated at 7-8 weeks old could be infected and died at 30 days post inoculation.The infection rate of another isolate TSWV-YN in A.thaliana Col-0 was also increased with the increase of plant age.This indicated that the infection rate of different TSWV isolates was affected by developmental age.In this study,two other tospoviruses,Euro/Asian-type TZSV and American-type INSV,were tested in Arabidopsis of different age.The results showed that the infection rates of these two viruses still increased with the development of Arabidopsis.Therefore,the infection rates of different types of tospoviruses in A.thaliana Col-0 were regulated by developmental stage.In addition,the infection of Cucumber mosaic virus(CMV)in A.thaliana at different ages was also tested.It was found that the infection of CMV on A.thaliana Col-0 was also affected by the developmental stage to some extent.Based on above results,this study further explored the developmental regulation mechanism of tospoviruses infection in Arabidopsis.For young Arabidopsis can not be infected by tospoviruses,one of the possible mechanism is the presence of young stage resistance to the virus.This study tested whether the antiviral pathway of RNA silencing was involved in this process.When TSWV-LE isolate was used to infect the Arabidopsis mutant dcl 2/3/4 or rdr 1/2/6 of different age,it was found that at different developmental stages,the infection rate of TSWV-LE in dcl 2/3/4 or rdr 1/2/6 was not different from that in wild type A.thaliana.These results suggest that even if there exists young stage resistance in Arabidopsis,the antiviral pathway of RNA silencing has nothing to do with the fact that young Arabidopsis is not susceptible to TSWV infection.Another possible mechanism is that the expression of susceptible host factors necessary for the virus infection is regulated by different developmental stages.The expression of susceptible host factors is low at young stage,and the virus is difficult to infect.With the increase of age,susceptible host factors follow,and the virus infection rate increased.Gene bombardment test showed that the efficiency of TSWV movement protein in 8-week-old Arabidopsis leaves was significantly higher than that in 4-week-old Arabidopsis leaves,suggesting that TSWV movement was indeed regulated by different developmental stages of plants.Pectin methylesterase and beta-1,3-glucanase are known to be important proteases involved in intercellular plasmodesmata permeability and have been shown to play an important role in intercellular movement of many plant viruses.In this study,the expression levels of pectin methylesterase and beta-1,3-glucanase in A.thaliana at 4 and 8 weeks old showed that the expression levels of these two genes were significantly higher in the 8 wk old plants than that in the 4 wk old Arabidopsis plants without TSWV infection.When TSWV infected,the expression of beta-1,3-glucanase involved in callose degradation was significantly up-regulated in 8-week-old Arabidopsis leaves.Although the expression of the gene was also up-regulated in TSWV infected 4-week-old Arabidopsis leaves,it was still significantly lower than that of TSWV uninfected 8-week-old Arabidopsis leaves.These results suggest that tospoviruses infection in Arabidopsis is regulated by different developmental stages,thus there may have developmentally regulated susceptible host factors in Arabidopsis.Maybe the interaction between these susceptible host factors and viruses determines the infection efficiency of tospoviruses in Arabidopsis.