Calcium-dependent protein kinases (CPKs) are plant proteins that directly bind calcium ions before phosphorylating substrates involved in biotic and abiotic stress responses, as well as development. Arabidopsis thaliana CPK3 (AtCPK3) is involved with plant signaling pathways such as stomatal movement regulation, salt stress response, apoptosis, seed germination and pathogen defense. In this study, AtCPK3 and its orthologues in relatively distant plant species such as rice (Oryza sativa, monocot) and kiwifruit (Actinidia chinensis, asterid eudicot) were analyzed in response to drought, bacteria, fungi, and virus infections. Two orthologues were studied in O. sativa, namely OsCPK1 and OsCPK15, while one orthologue—AcCPK16—was identified in A. chinensis. Reverse-transcriptase quantitative PCR (RT-qPCR) analysis revealed that OsCPK1 and AcCPK16 exhibit similar responses to stressors to AtCPK3. OsCPK15 responded differently, particularly in bacterial and fungal infections. An increase in expression was consistently observed among AtCPK3 and its orthologues in response to virus infection. Overexpression mutants in both Arabidopsis and kiwifruit showed slight tolerance to drought, while knockout mutants were slightly more susceptible or had little difference with wild-type plants. Overexpression mutants in Arabidopsis showed slight tolerance to virus infection. These findings highlight the importance of AtCPK3 and its orthologues in drought and pathogen responses and suggest such function must be conserved in its orthologues in a wide range of plants.

To our knowledge, there are no reports that demonstrate the use of host molecular markers for the purpose of detecting generic plant virus infection. Two approaches involving molecular indicators of virus infection in the model plant Arabidopsis thaliana were examined: the accumulation of small RNAs (sRNAs) using a microfluidics-based method (Bioanalyzer); and the transcript accumulation of virus-response related host plant genes, suppressor of gene silencing 3 (AtSGS3) and calcium-dependent protein kinase 3 (AtCPK3) by reverse transcriptase-quantitative PCR (RT-qPCR). The microfluidics approach using sRNA chips has previously demonstrated good linearity and good reproducibility, both within and between chips. Good limits of detection have been demonstrated from two-fold 10-point serial dilution regression to 0.1 ng of RNA. The ratio of small RNA (sRNA) to ribosomal RNA (rRNA), as a proportion of averaged mock-inoculation, correlated with known virus infection to a high degree of certainty. AtSGS3 transcript decreased between 14- and 28-days post inoculation (dpi) for all viruses investigated, while AtCPK3 transcript increased between 14 and 28 dpi for all viruses. A combination of these two molecular approaches may be useful for assessment of virus-infection of samples without the need for diagnosis of specific virus infection.

Lettuce necrotic yellows virus is a type of species in the Cytorhabdovirus genus and appears to be endemic to Australia and Aotearoa New Zealand (NZ). The population of lettuce necrotic yellows virus (LNYV) is made up of two subgroups, SI and SII. Previous studies demonstrated that SII appears to be outcompeting SI and suggested that SII may have greater vector transmission efficiency and/or higher replication rate in its host plant or insect vector. Rhabdovirus glycoproteins are important for virus–insect interactions. Here, we present an analysis of LNYV glycoprotein sequences to identify key features and variations that may cause SII to interact with its aphid vector with greater efficiency than SI. Phylogenetic analysis of glycoprotein sequences from NZ isolates confirmed the existence of two subgroups within the NZ LNYV population, while predicted 3D structures revealed the LNYV glycoproteins have domain architectures similar to Vesicular Stomatitis Virus (VSV). Importantly, changing amino acids at positions 244 and 247 of the post-fusion form of the LNYV glycoprotein altered the predicted structure of Domain III, glycosylation at N248 and the overall stability of the protein. These data support the glycoprotein as having a role in the population differences of LNYV observed between Australia and New Zealand.