Virus-Induced Gene Silencing Using Tobacco Rattle Virus as a Tool to Study the Interaction between <i>Nicotiana attenuata</i> and <i>Rhizophagus irregularis</i>

<div><p>Most land plants live in a symbiotic association with arbuscular mycorrhizal fungi (AMF) that belong to the phylum Glomeromycota. Although a number of plant genes involved in the plant-AMF interactions have been identified by analyzing mutants, the ability to rapidly manipulate gene expression to study the potential functions of new candidate genes remains unrealized. We analyzed changes in gene expression of wild tobacco roots (<i>Nicotiana attenuata</i>) after infection with mycorrhizal fungi (<i>Rhizophagus irregularis</i>) by serial analysis of gene expression (SuperSAGE) combined with next generation sequencing, and established a virus-induced gene-silencing protocol to study the function of candidate genes in the interaction. From 92,434 SuperSAGE Tag sequences, 32,808 (35%) matched with our in-house <i>Nicotiana attenuata</i> transcriptome database and 3,698 (4%) matched to <i>Rhizophagus</i> genes. In total, 11,194 Tags showed a significant change in expression (p<0.05, >2-fold change) after infection. When comparing the functions of highly up-regulated annotated Tags in this study with those of two previous large-scale gene expression studies, 18 gene functions were found to be up-regulated in all three studies mainly playing roles related to phytohormone metabolism, catabolism and defense. To validate the function of identified candidate genes, we used the technique of virus-induced gene silencing (VIGS) to silence the expression of three putative <i>N</i>. <i>attenuata</i> genes: <i>germin-like protein</i>, <i>indole-3-acetic acid-amido synthetase GH3</i>.<i>9</i> and, as a proof-of-principle, <i>calcium and calmodulin-dependent protein kinase</i> (<i>CCaMK)</i>. The silencing of the three plant genes in roots was successful, but only <i>CCaMK</i> silencing had a significant effect on the interaction with <i>R</i>. <i>irregularis</i>. Interestingly, when a highly activated inoculum was used for plant inoculation, the effect of <i>CCaMK</i> silencing on fungal colonization was masked, probably due to trans-complementation. This study demonstrates that large-scale gene expression studies across different species induce of a core set of genes of similar functions. However, additional factors seem to influence the overall pattern of gene expression, resulting in high variability among independent studies with different hosts. We conclude that VIGS is a powerful tool with which to investigate the function of genes involved in plant-AMF interactions but that inoculum strength can strongly influence the outcome of the interaction.</p>