PSTVd Symptom Induction Mechanism On Nature Host Potato

Viroids are small non-coding RNAs whose replication induce diseases in a variety of plant species.Potato spindle tuber viroid(PSTVd)is the first discovered and the most studied viroid.Potato(Solanum tuberosum L.)is the natural host of PSTVd where infection results in stunting and distortion of leaves and tubers.PSTVd has been reported in many countries and regions,seriously affecting potato yield and quality,and the molecular mechanism(s)responsible for these effects remain poorly understood.Replication of PSTVd is accompanied by the production of a complex array of small RNAs that are thought to play an important role in symptom induction.Here we report that PSTVd sRNAs direct RNA silencing in potato against StTCP23,member of the TCP(teosinte branched1/Cycloidea/ Proliferating cell factor)transcription factor family genes that play an important role in plant growth and development as well as hormonal regulation,especially in responses to gibberellic acid(GA).The StTCP23 transcript has 21-nucleotide sequence complementarity in its 3' untranslated region with the virulence-modulating region(VMR)of PSTVd strain RG1,and was downregulated in PSTVd-infected potato plants.Analysis using 3' RNA ligase-mediated rapid amplification of cDNA ends(3' RLM RACE)confirmed cleavage of StTCP23 transcript at the expected sites within the complementarity with VMR-derived sRNAs.Expression of these VMR sRNA sequences as artificial miRNAs(amiRNAs),transgenic potato plants resulted in phenotypes reminiscent of PSTVd-RG1-infected plants.Furthermore,the severity of the phenotypes displayed was correlated with the level of amiRNA accumulation and the degree of amiRNA-directed down-regulation of StTCP23.In addition,virus-induced gene silencing(VIGS)silencing of StTCP23 in potato using virus-induced gene silencing also resulted in PSTVd-like phenotypes.Members of the TCP family of transcription factors play an important role in plant growth and development as well as hormonal regulation,especially in the gibberellic acid(GA)pathway.We systematically analyzed the evolutionary history and hormone regulation of 23 members of the potato TCP gene family.There is a high degree of homology between the potato StTCPs and tomato SlTCPs,and the target gene StTCP23 shows the highest homology with tomato SlTCP23 and Arabidopsis AtTCP14/15.The results of RNA-seq and qRT-PCR analyses indicate that StTCP23 is widely expressed in many tissues.Expression levels are especially high in dormant tubers,stolon,stems and nodes,suggests that they may playing important role in potato growth,stem branching and tuber development.Our data also demonstrate that the expression of several StTCP genes showed dramatic changes under GA treatments,suggesting that these genes may have functions in the regulation of GA mediated plant growth and development.Consistent with the function of TCP family genes,these amiRNA lines with silenced StTCP23 expression showed a decrease in GA levels as well as alterations to the expression of GA biosynthesis and signaling genes StGA3ox2,StGA20ox1,StGA2ox1,StGA7 ox,DELLA-GAI,and GID1,which previously implicated in tuber development.The effects of exogenous gibberellin application further confirmed that this hormone plays an important regulatory role in the potato tuber formation.Treatment with the gibberellin inhibitor Paclobutrazol(PBZ)resulted in an increased number of tubers,almost all of which were small in size and abnormal in shape.This abnormal phenotype could be reversed by treatment with exogenous GA.Taken together,our results indicate that sRNAs derived from the VMR of PSTVd-RG1 direct silencing of StTCP23 expression,thereby disrupting the signaling pathways regulating GA metabolism,leading to plant stunting and formation of spindle-shaped tubers.