Cell Fractionation and the Identification of Host Proteins Involved in Plant-Virus Interactions.

Plant viruses depend on host cellular factors for their replication and movement. There are cellular proteins that change their localization and/or expression and have a proviral role or antiviral activity and interact with or target viral proteins. Identification of those proteins and their roles during infection is crucial for understanding plant-virus interactions and to design antiviral resistance in crops. Important host proteins have been identified using approaches such as tag-dependent immunoprecipitation or yeast two hybridization that require cloning individual proteins or the entire virus. However, the number of possible interactions between host and viral proteins is immense. Therefore, an alternative method is needed for proteome-wide identification of host proteins involved in host-virus interactions. Here, we present cell fractionation coupled with mass spectrometry as an option to identify protein-protein interactions between viruses and their hosts. This approach involves separating subcellular organelles using differential and/or gradient centrifugation from virus-free and virus-infected cells (1) followed by comparative analysis of the proteomic profiles obtained for each subcellular organelle via mass spectrometry (2). After biological validation, prospect host proteins with proviral or antiviral roles can be subject to fundamental studies in the context of basic biology to shed light on both virus replication and cellular processes. They can also be targeted via gene editing to develop virus-resistant crops.

Induction and suppression of gene silencing in plants by nonviral microbes.

Gene silencing is a conserved mechanism in eukaryotes that dynamically regulates gene expression. In plants, gene silencing is critical for development and for maintenance of genome integrity. Additionally, it is a critical component of antiviral defence in plants, nematodes, insects, and fungi. To overcome gene silencing, viruses encode effectors that suppress gene silencing. A growing body of evidence shows that gene silencing and suppression of silencing are also used by plants during their interaction with nonviral pathogens such as fungi, oomycetes, and bacteria. Plant-pathogen interactions involve trans-kingdom movement of small RNAs into the pathogens to alter the function of genes required for their development and virulence. In turn, plant-associated pathogenic and nonpathogenic microbes also produce small RNAs that move trans-kingdom into host plants to disrupt pathogen defence through silencing of plant genes. The mechanisms by which these small RNAs move from the microbe to the plant remain poorly understood. In this review, we examine the roles of trans-kingdom small RNAs and silencing suppressors produced by nonviral microbes in inducing and suppressing gene silencing in plants. The emerging model is that gene silencing and suppression of silencing play critical roles in the interactions between plants and their associated nonviral microbes.