Functional analysis of NbCPIP2 in nicotiana benthamiana and related gene expression profiling of chrysanthemum in response to potato virus X infection

Abstract

Protein interactions between viruses and host plants are important for the successful viral life cycle because viruses contain a limited numbers of viral genes resulting in relying on the host machinery. In current study, I characterized two homologous host proteins (NbCPIP2a and NbCPIP2b) derived from a previous study which identified several host proteins interacting with potato virus X (PVX) RNAs by Northwestern blot analysis. I confirmed that two proteins interacted with stem-loop 1 (SL1) RNA structure and capsid protein of PVX. Agrobacterium-mediated transient assay using Nicotiana benthamiana showed that overexpression of each protein affected replication and movement of PVX in the inoculated leaves positively. Overexpression of NbCPIP2a positively affected the systemic movement of PVX whereas overexpression of NbCPIP2b did not show any effect on systemic movement of PVX. Transient silencing experiments demonstrated that replication and movement of PVX were reduced in NbCPIP2a and NbCPIP2b-silenced leaves. However, PVX systemic movement in the entire plant was not changed after silencing of NbCPIP2a and NbCPIP2b genes, respectively. Although the two proteins are associated with plasma membranes, PVX infection did not affect their subcellular localization. As a result, NbCPIP2a and NbCPIP2b, which differ in five amino acids, specifically bind to PVX SL1 RNAs as well as to CP

however, their detailed functional roles were somehow different although two proteins share redundant functions. It is important to identify host factors associated with virus infection. Moreover, the functional roles of host proteins with respective virus might be dependent on host plants. Therefore, it might be of interest to find additional host factors in other non-model plants. For that, I used chrysanthemum plants, which are susceptible to many viruses and viroids. Based on previous expressed sequence tags of chrysanthemum, I established a chrysanthemum microarray system for the first time. I carried out transcriptome analysis of chrysanthemum in response to three different viruses including cucumber mosaic virus (CMV), tomato spotted wilt virus (TSWV) and PVX. A chrysanthemum135K microarray was used for the gene expression profiles of the chrysanthemum at the early stage of virus infection. As a result, a total of 125, 70, and 124 differentially expressed genes (DEGs) have been identified for CMV, TSWV and PVX, respectively. Although most identified DEGs were virus specific, I identified 33 DEGs, which were commonly regulated by three viruses. Using gene ontology (GO) enrichment analysis, 132 GO terms were significantly identified. Of identified GO terms, stress response and MCM complex were frequently identified for three viruses. I found that many genes are involved in stress responses including chitin response and ethylene mediated signaling pathway. Their gene expression was strongly induced. This result suggests that the stress associated genes play critical roles for host innate immune system. It was also found that TSWV infection led to down-regulation of genes involved in DNA metabolic process such as DNA replication, chromatin organization, histone modification and cytokinesis. In summary, I found many important biological process associated with infection of three plant viruses and provides potential host genes which might be involved in plant resistant against virus infection.