Differential resistance to Citrus psorosis virus in transgenic Nicotiana benthamiana plants expressing hairpin RNA derived from the coat protein and 54K protein genes

Abstract

Citrus psorosis virus (CPsV), genus Ophiovirus, family Ophioviridae, is the causal agent of a serious disease affecting citrus trees in many countries. The viral genome consists of three ssRNAs of negative polarity. Post-transcriptional gene silencing (PTGS), a mechanism of plant defence against viruses, can be induced by transgenic expression of virus-derived sequences encoding hairpin RNAs. Since the production of transgenic citrus lines and their evaluation would take years, a herbaceous model plant, Nicotiana benthamiana, was used to test hairpin constructs. The expression of self-complementary hairpin RNA fragments from the coat protein (cp) and 54K genes of the Argentine CPsV 90-1-1 isolate conferred resistance on N. benthamiana plants, indicating that these constructs are good candidates for the transformation of citrus plants. The degree of resistance obtained varied depending on the viral sequence chosen. The analysis of the levels of small interfering RNA accumulation and viral RNAs indicated that the construct derived from cp gene was better at inducing PTGS than that originating from the 54K gene. The dependence of PTGS induction on the degree of identity between the target and the inducer transgene sequences was tested using sequences derived from CPV4, a more distant isolate of CPsV, as PTGS targets. Efficient silencing induction was also obtained to this isolate through the expression of the cp-derived hairpin. This is the first report of transgenic-resistant plants within the context of this serious citrus disease.

Resistance to Citrus psorosis virus in transgenic sweet orange plants is triggered by coat protein–RNA silencing

“…RT-PCR was performed with the primers D/C1 from cp gene or A/B from 54k gene (680 and 369-bp fragment respectively, see Table 1) as described previously (Reyes et al, 2009). …”

“…Lengths of hairpin-CP (ihpCP) hairpin-54K (ihp54K) and hairpin-24K (ihp24K) fragments are 372, 436, and 312 bp respectively. ihpCP and ihp54K constructs were generated previously (Reyes et al, 2009).…”

Recent advances in Citrus psorosis virus

“…Several citrus species have been transformed with the CPG of a severe strain of CPsV but presently there is little published information on the resistance of such plants to CPsV infection [23,48,49,50,81]. The same strategy was used by Zanek et al [81] producing 21 independent lines of transgenic sweet orange (CP-lines) expressing low and variable amounts of CPsV CP (isolate CPV-4).…”

“…Recently, Reyes et al [48] showed that the resistance mechanism is the post transcriptional gene silencing (PTGS). A mechanism of RNA degradation involved in plant protection against viruses and can be induced by transgenic expression of pathogen-derived sequences encoding hairpin RNAs.…”

“…Efficient silencing induction was also obtained to this isolate through the expression of the CP-derived hairpin. Furthermore, alignment between CPV-4 and the RNA2-hairpin fragment (54K-fragm) of CPsV 90-1-1 sharing 81 % identity, a good degree of gene silencing is achieved by means of genes sequences having less than 100 % identity between the inducer siRNA segment and the target sequence making unlikely the explanation of the limited resistance observed in these intronhairpin-54K (ihp54K) lines by means of lack of sequence identity between isolates [48].…”