Expression of artificial microRNAs in tomato confers efficient and stable virus resistance in a cell-autonomous manner

Abstract

Expression of artificial microRNAs (amiRNAs) in plants can target and degrade the invading viral RNA, consequently conferring virus resistance. Two amiRNAs, targeting the coding sequence shared by the 2a and 2b genes and the highly conserved 3' untranslated region (UTR) of Cucumber mosaic virus (CMV), respectively, were generated and introduced into the susceptible tomato. The transgenic tomato plants expressing amiRNAs displayed effective resistance to CMV infection and CMV mixed with non-targeted viruses, including tobacco mosaic virus and tomato yellow leaf curl virus. A series of grafting assays indicate scions originated from the transgenic tomato plant maintain stable resistance to CMV infection after grafted onto a CMV-infected rootstock. However, the grafting assay also suggests that the amiRNA-mediated resistance acts in a cell-autonomous manner and the amiRNA signal cannot be transmitted over long distances through the vascular system. Moreover, transgenic plants expressing amiRNA targeting the 2a and 2b viral genes displayed slightly more effective to repress CMV RNA accumulation than transgenic plants expressing amiRNA targeting the 3' UTR of viral genome did. Our work provides new evidence of the use of amiRNAs as an effective approach to engineer viral resistance in the tomato and possibly in other crops.

Stability of Transgenic Resistance Against Plant Viruses

“…Recent advances like the construction of chimeric IR constructs incorporating sequences derived from different virus species if combined with epidemiological data and pest risk analyses could reduce the effect of mixed virus infections on the resistance (Bucher et al, 2006;Dafny-Yelin & Tzfira, 2007;Kung et al, 2009). Recently, virus resistance was achieved through expression of amiRNAS against viral coding sequences (Ding & Voinnet, 2007;Duan et al, 2008;Niu et al, 2006;Qu et al, 2007;Simon-Mateo & Antonio Garcia, 2006;Zhang et al, 2011). Although there was evidence that amiRNA-mediated virus resistance may not be inhibited by low temperature (Niu et al, 2006) this possibly depends on the plant species examined (Qu et al, 2007).…”

“…This strategy resulted in a high frequency of produced resistant plants. A most recent approach used modified plant miRNA cistrons to produce a range of antiviral artificial miRNAs (amiRNAs) (Niu et al, 2006;Qu et al, 2007;Schwab et al, 2006;Simon-Mateo & Antonio Garcıa, 2007;Zhang et al, 2011).…”

Artificial miRNAs for Specific Gene Silencing and Engineering Virus Resistance in Plants

“…In plants, amiRNAs have been successfully used for downregulating endogenous genes [25,29,31] and developing transgenic virus resistance against turnip yellow mosaic virus (TYMV), turnip mosaic virus (TuMV) in Arabidopsis [31]; tomato leaf curl virus [34]; cucumber mosaic virus (CMV)in tobacco [35]; CMV in tomato [36] and cassava brown streak virus (CBSV) and Cassava brown streak Uganda virus (CBSUV) in cassava [37].…”

Small Non-Coding RNAs in Plant Immunity

“…Indeed several studies demonstrated that artificial miRNAs (amiRNAs) targeting key components of the viral replication machinery can efficiently impair viral growth upon infection [59][60][61][62][63][64][65]. These efforts have revealed that such resistance is cell-autonomous, inheritable, more efficient than siRNA-mediated strategies and successful in blocking viral replication and movement [59,66]. Furthermore transgenic plants expressing dimeric or polycistronic amiRNAs directed against different viruses result in a wider spectrum of viral resistance [60,61,63].…”