Functional Research On The Resistance Of Tomato SlRDR1 To Citrus Exocortis Viroid

Viroids are composed by a small,non-protein coding,circular,single-stranded RNA(ss RNA)with compact secondary structure.As the smallest plant pathogen,viroids can infect plant cells and cause serious diseases on economically important crops and ornamental plants such as citrus,peach,potato,and tomato.CEVd infects most citrus species,tomatoes,and eggplants,and has a significant impact on plant production,especially the breeding of virus-free citrus seedlings.Although previous studies on viroids interactions with plants have been done,the defense mechanism of plants against CEVd is not clear.In this study,we used RNA-dependent RNA polymerase 1(RDR1),a key component of the RNA silencing pathway,as an entry point to alter the expression of SlRDR1 by VIGS technology and transgenic technology,analyzed the effects of changes in SlRDR1 expression on viroid accumulation,and preliminarily explored the role of SlRDRs in resistance to CEVd infection and the interactions between SlRDRs.The main findings of this paper are as follows.1.Viroid and phytohormones induce RDR1 expressionCEVd induced up-regulation expression of SlRDR1 and CmRDR1 genes.The expression of SlRDR1 was most significantly up-regulated in the pre-and mid-infection period of CEVd,and the expression of SlRDR1 was nearly 8-fold higher than that of control plants at 14 dpi.Expression of CmRDR1 in CEVd-infected plants was more than four times higher than that of the control plants at 4 mpi.Different strains of CEVd(CEVd-A,CEVd-DE30 and CEVd-MS9)induced SlRDR1 expression in tomato,and CEVd-A induced the highest expression of SlRDR1 gene.In this study,tomato leaves were sprayed with 2 mM SA,100 μM JA,100 μM Me JA,100 μM GA,100 μM ABA,5mM ETH and 100 μM anhydrous ethanol(control),and the expression of SlRDR1 gene was examined at 3 h,6 h,9 h,12 h,24 h and 48 h after spraying.The results showed that SlRDR1 in the SA-treated group reached the highest expression at 24 h,more than four times that of the control group.In addition,the SlRDR1 gene in tomato was tissue-specific,with the highest expression in leaves,especially older leaves,followed by younger leaves.2.SlRDR1 is involved in defence against CEVdIn order to clarify the role of RDR1 in the process of viroid infection,we reduced the expression of SlRDR1 gene by silencing it using VIGS technique,then observed the plant phenotype after inoculation with CEVd and measured the accumulation of CEVd genomic RNA.The TRV-based VIGS vectors were constructed and transformed into Agrobacterium and then infected with tomatoes and inoculated with CEVd at 14 days.The results showed that TRV:SlRDR1 plants were more susceptible to CEVd infection and showed more pronounced leaf curling symptoms,and the corresponding CEVd genomic RNA accumulation was significantly higher than that of the control plants(TRV:00).In addition,in order to further clarifying the role of the SlRDR1 gene in resisting viroid infection,the SlRDR1 gene was overexpressed on tomatoes through transgenic technology.The plant symptom was observed and the accumulation of CEVd genomic RNA was detected in CEVd-infected transgenic plants.The results showed that SlRDR1 transgenic tomato plants were able to delay the accumulation of CEVd genomic RNA in the early stages of infection,with a lower accumulation of CEVd compared to EV transgenic plants.However,there was no significant difference in CEVd accumulation between the two at 28 dpi.The SlRDR1 transgenic plants showed no obvious symptoms,but the EV transgenic plants showed leaf curling and slight dwarfing symptoms.3.Complex silencing of SlRDRs significantly enhances susceptibility of tomato to CEVd infectionWhen exploring the induction of SlRDRs gene expression by CEVd,it was found that SlRDR2,SlRDR6 a,and SlRDR6 b genes can also respond to CEVd infection.Therefore,it is speculated that in addition to SlRDR1,SlRDR2,SlRDR6 a,and SlRDR6 b genes also play a role in resisting CEVd infection in tomatoes.To verify this hypothesis,VIGS recombinant vectors containing SlRDR2,SlRDR6 a,and SlRDR6 b genes were constructed,transformed into Agrobacterium,and infected with tomatoes.CEVd was inoculated 14 days after infection.Similar to SlRDR1,all the treated plants showed greater sensitivity to CEVd infection: compared to the control,the treated plants had significantly higher accumulation of CEVd genomic RNA and more pronounced leaf curling symptoms.In other words,SlRDR2,SlRDR6 a,and SlRDR6 b genes have a role in defending against CEVd infection.To research the role between SlRDRs,multiple SlRDRs genes were silenced simultaneously in tomato plants using VIGS technique.Then plant symptom was observed and the accumulation of CEVd was detected after CEVd infection.The results showed that when two SlRDRs genes were silenced,the sensitivity of tomato plants to CEVd infection was not significantly different from that of SlRDRs single gene silenced plants.When three SlRDRs genes are silenced,the sensitivity of tomato to CEVd infection is enhanced.When four SlRDRs genes were silenced,the sensitivity of tomatoes to CEVd infection was significantly enhanced,and tomato plants exhibited severe dwarfing symptoms.The results showed that the susceptibility of tomato to CEVd gradually increased as the number of genes silenced simultaneously increased.The SlRDRs gene may jointly resist the infection of CEVd in tomato plants,and when individual genes are silenced,other SlRDRs play a defensive role against CEVd through gene complementation.When the SlRDRs genes with disease resistance were silenced,tomato plants showed significant sensitivity to CEVd infection.