Molecular Mechanism Of The Vertical Transmission Of Rice Stripe Virus Mediated By Dopamine In Laodelphax Striatellus

Laodelphax striatellus is one of the important rice pests,which not only causes damage by stinging rice but also acts as the vector of Rice stripe virus（RSV）by the way of horizontal and vertical transmission.Rice stripe disease caused by RSV is one of the most devastating plant viruses of rice and causes enormous losses in production,known as "Rice Cancer".RSV has to overcome multiple barriers in the processes of infection,replication and transmission in the vector insects,and a series of studies have been carried out on the mechanisms of RSV crossing the transmission barriers of midgut,salivary gland and ovary in L.striatellus.Although there are some reports about the involvement of neural signallings in plant virus transmission,the underlying molecular mechanism remains unknown.Dopamine（DA）is an important biogenic amine that is widely distributed in vertebrates and invertebrates,which is involved in modulating multiple behavioral and physiological processes,such as development,olfactory,memory and learning.Previous studies indicated that RSV vertical transmission rates were significantly induced by DA in the viruliferous L.striatellus.However,the mechanism of DA in the transmission of RSV is still unclear.The actions of DA have been mediated through binding to their specific dopamine receptor,which belongs to G protein-coupled receptor.In this study,we cloned four dopamine receptors from L.striatellus.,characterized the expression patterns of these four receptors at different developmental stages and in various tissues of L.striatellus by qRT-PCR,and investigated the roles of DA in regulating RSV transmission by DA injection and RNAi in the viruliferous L.striatellus.Dopamine receptors are important potential targets of insecticides,this study will provide fundamental information for controlling vector insects and blocking the transmission of plant virus.1.Gene cloning and expression patterns of dopamine receptors in L.striatellusWe identified and cloned four dopamine receptors,which were named DOP1,DOP2,DOP3 and DopEcR,respectively.Transmembrane prediction showed that these dopamine receptors display the typical characteristics of G protein-coupled receptors that contains seven transmembrane domains（TM）.Multiple sequence alignments showed that the homology between the dopamine receptors of L.striatellus and other insect homologous receptors were very high,with 60%-98%identity.The expression patterns of four dopamine receptors were analyzed by qRT-PCR.The results indicated that the expressions of dopamine receptors could be detected at the different development stages of L.striatellus.Dopamine receptors were highly expressed in the eggs,and expression levels gradually decreased with age stages.Tissue expression analysis showed that four dopamine receptors have relatively high expressions in the brain of L.striatellus,and the expression profiles of DOP1,DOP2,DOP3 and DopEcR were different in various tissues.2.Functional characterization of dopamine receptors in regulating RSV transmission in the viruliferous L.striatellusIn order to further elucidate the molecular mechanism of DA in regulating RSV transmission,we used DA injection and RNAi.The results showed RSV vertical transmission rates were significantly induced by DA injection.The relative expression of capsid protein（CP）and viral RNA3 segment（RNA3）were also significantly increased in the viruliferous L.striatellus.Comparing the expression levels of dopamine receptors in the non-viruliferous and viruliferous L.striatellus suggested that dopamine receptors may regulate RSV transmission.Knockdown of dopamine receptors using RNAi preliminarily confirmed that DOP2 and DOP3 may play important roles in regulating RSV transmission.Compared with the dsGFP group,the injection of dsDOP2 and dsDOP3 decreased RSV accumulations in the viruliferous L.striatellus.Vertical transmission rates of the dsDOP2-injected and dsDOP3-injected groups were significantly decreased.Knockdown of DOP2 and DOP3 significantly reduced the relative expression levels of Vg and Vg receptor（VgR）.However,silencing DOP3 resulted in the decline of fecundity and silencing DOP2 did not.In addition,qRT-PCR results verified that DA induced the relative expression levels of Vg and VgR.