Functional Analysis Of NbALD1 And Nbe-miR166h-p5 In Viral Infection

Plant virus is a kind of important plant pathogen,causing serious disease on crop,with huge economic loss.During the process of viral infection,various resistant pathways would act to resist virus infection and attenuate viral symptom.It is significantly important to investigate the resistance functioning in viral infection to understand plant and virus interaction and offer new strategies to resist virus infection.Based on early analysis of miRNA transcriptome profile after PVX and TuMV infected Nicotiana benthamiana,we found that gene NbALD1(AGD2-like defense response protein 1)and miR166h-p5(nbe-miR166h-p5)were up-regulated by virus infection.In this study,we deeply investigated the role of NbALD1 and nbe-miR166h-p5 in virus infection.ALD1 encodes an aminotransferase,which plays an important role in the synthesis of pipecolic acid(Pip).It has been reported that Pip is a key factor in systemic acquired resistance(SAR).Ald1 mutant plants were more susceptible to P.s.tomato DC3000 compared to wild-type plants,while ALD1-overexpressed Arabidopsis showed more resistance to P.s.tomato DC3000 and overexpression of ALD1 in rice plant enhanced plants'resistance to Magnaporthe oryzae.In this study,qRT-PCR confirmed that NbALD1 was up-regulated after TuMV infected.NbALD1 was silenced by tobacco rattle virus(TRV)-induced gene silencing(VIGS)system to investigate the role of NbALD1.ALD1 silence didn t change the morphological phenotypes of N.benthamiana.However,silencing of NbALD1 resulted in more viral accumulation in N.benthamiana,along with faster systemic infection rate.Exogenous application of pipecolic acid improved the resistance of wild-type and NbALD1-silenced plants to TuMV infection.In addition,NbALDl-silenced plants were more susceptible to PVX and TMV infection.All together,silenced NbALDl was beneficial to the infection of TuMV.PVX and TMV.Further research showed that salicylic acid and NPR1(the downstream gene of salicylic acid signal pathway)was down-regulated in NbALD1-silenced plants.Exogenous application of pipecolic acid partially compromised susceptibility of NahG transplants to TuMV,which indicating that the susceptibility to TuMV caused by NbALD1 silence was dependent just on down-regulated salicylic acid,but also some other pathways.What's more,NbALD1 silence increased the content of ethylene.Suppress ethylene related genes such as ACS1,ACO1 and EIN2 enhanced the resistance to TuMV,exogenous application of AVG also increase desease resistance,while exogenous application of ACC induced TuMV infection.Therefore,we infer that NbALD1 silence may increase susceptibility to TuMV through accumulated ethylene.Taken together,NbALD1 silence has a positive regulation on salicylic acid signal pathway,and negative regulation on ethylene signal pathway to participate in virus resistance.Research increasing suggests that miRNA plays an important role in virus infection.Here,we analyzed the profile of miRNAs after PVX infected Nicotiana benthamiana,and found that nbe-miR166h-p5,located on the 5'-terminal of miR166h precursor was induced after PVX infected.We tried to explore its role in virus infection.Using TRV-based miRNAs suppression,we found that suppression of nbe-miR166h-p5 caused leaves dark green with increased chlorophyll.And its suppression attenuated the yellowing symptom caused by PVX,along with decreased PVX accumulation.nbe-miR166h-p5 was also upregulated by TuMV and PMMoV,and its suppression also attenuated leaf yellowing symptom and decreased viral accumulation.And we predicted and indentified three targets of nbe-miR166h-p5.In a word,our results demonstrated the association of nbe-miR166h-p5 with symptoms of PVX,TuMV and PVX,proving evidences for disturbed miRNAs expression participating in viral symptom.Taken together,our study re'vealed that NbALD1 participate in the process of plant resistance to virus through positively regulating of salicylic acid signal pathway and negatively regulating of ethylene signal pathway,nbe-miR166h-p5 take part in the symptoms of plant virus and anti-virus infection through altered expression level.This study provides us a further insight to investigate how the disease-related components of the plant involved in the process of viral infection.