Cucumber mosaic virus: virus movement, resistance, disease symptoms and suppression of gene silencing

Abstract

Dysfunctional viral movement protein (MP) genes have been shown to give broad spectrum resistance when expressed in transgenic plants. Transgenic tobacco lines were generated expressing dysfunctional MP of Cucumber mosaic virus (CMV) strain Fny, modified by polymerase chain reaction-site directed mutagenesis. The transgenic lines were molecularly analysed, and challenged with CMV isolates belonging to subgroups I and II. However, unexpectedly none of the transgenic MP lines conferred resistance, irrespective of transgene copy number and of the accumulation of putative dysfunctional MP. In addition, hybrid lines expressing two different modified MPs were also susceptible to CMV infection.As a first step towards studying the mechanism of post-transcriptional gene silencing (PTGS)-based immunity to Potato virus Y (PVY) and its suppression by CMV, accumulation pattern of CMV and PVY were studied in infected tobacco plants. CMV was detected in all leaves, but concentrated in young leaves. On the other hand, PVY mostly accumulated in older systemic leaves. Dual infection of CMV and PVY benefited both viruses, resulting in higher virus titres and more severe symptoms. CMV aided PVY movement to the top part of the plant.CMV and PVY co-inoculation of PVY-immune transgenic tobacco plants, expressing a sense- or a dsRNA-transgene, showed that CMV induced the breakage of PTGS-based immunity to PVY. Increased time intervals between CMV and subsequent PVY inoculations resulted in an increasing proportion of initially immune plants becoming infected with PVY. Immunity was broken in 100% of initially immune transgenic plants when PVY was inoculated on a leaf newly emerged following CMV inoculation. This confirmed the proposed mode of action of PTGS suppression by CMV 2b protein through preventing the initiation of PTGS. Interestingly, PVY failed to establish a long term systemic infection, despite the initial immunity breakdown and continued presence of CMV in all leaves. Attempts to re-inoculate PVY on the youngest leaves failed indicating a highly resistant state of these transgenic plants in the presence of CMV.Molecular information on Australian CMV isolates is limited. Six Australian CMV isolates were characterised to determine their experimental host range, disease symptoms, and evolutionary relatedness. Host differentials and differentiating symptoms were identified for each isolate. Phylogenetic analyses based on the sequences of RNA 3, and sequence analyses of the 2b gene placed the isolates into the known 3 CMV subgroups.The CMV 2b protein is involved in virus movement, pathogenicity, and suppression of plant defence. Two mutants were generated by inserting a stop codon in the 2b and putative 2c genes of Fny-CMV. The CMV 2b mutant infected tobacco plants and moved systemically, but did not cause symptoms, indicating that CMV 2b protein is involved in CMV symptom development. CMV subgroup I isolates encode a putative CMV 2c gene that may function as a modulator of symptom severity, because a 2c mutant had significantly increased symptom severity.To determine whether the 2b gene or its product are involved in the development of disease symptoms and host range specificity, this gene was cloned from five Australian isolates, and moved into the Q-CMV background (Q-CMV RNAs 1 and 3, plus recombinant RNA 2). The 2b genes from Australian CMV isolates which showed distinct host range and disease symptoms in Nicotiana species, sweetcom, tomato, and capsicum, were cloned in an infectious RNA 2 clone of Q-CMV. Inoculation of Nicotiana species and capsicum with recombinant hybrid RNA 2 Q-CMV carrying 2b derived from severe isolates changed Q-CMV symptoms from mild to severe. On the other hand, recombinant Q-CMV carrying 2b derived from mild isolates maintained the mild Q-CMV symptoms. Tomato infected by recombinant hybrid RNA 2 Q-CMV did not show symptoms. However, none of the recombinant hybrid RNA 2 Q-CMV infected sweetcom, which indicates that 2b is not sufficient to overcome CMV resistance in sweet com. These results indicate that the 2b protein is a determining factor in the severity of disease symptoms in a host specific manner. Diagnostic RT- PCR restriction fragment assays were developed to confirm the source of 2b gene in the recombinant Q-CMV.