Viruses have different strategies for infecting their hosts. Fast and acute infections result in the development of severe symptoms and may cause the death of the plant. By contrast, in a persistent interaction, the virus can survive within its host for a long time, inducing only mild symptoms. In this study, we investigated the gene expression changes induced in CymRSV-, crTMV-, and TCV-infected Nicotiana benthamiana and in PVX- and TMV-U1-infected Solanum lycopersicum plants after the systemic spread of the virus by two different high-throughput methods: microarray hybridization or RNA sequencing. Using these techniques, we were able to clearly differentiate between acute and persistent infections. We validated the gene expression changes of selected genes by Northern blot hybridization or by qRT-PCR. We show that, in contrast to persistent infections, the drastic shut-off of housekeeping genes, downregulation of photosynthesis-related transcripts and induction of stress genes are specific outcomes with acute infections. We also show that these changes are not a consequence of host necrosis or the presence of a viral silencing suppressor. Thermal imaging data and chlorophyll fluorescence measurements correlated very well with the molecular changes. We believe that the molecular and physiological changes detected during acute infections mostly contribute to virus symptom development. The observed characteristic physiological changes associated with economically more dangerous acute infections could serve as a basis for the elaboration of remote monitoring systems suitable for detecting developing virus infections in crops. Moreover, as molecular and physiological changes are characteristics of different types of virus lifestyles, this knowledge can support risk assessments of recently described novel viruses.

Woody perennial plants like grapevine and fruit trees can be infected by several viruses even as multiple infections. Since they are propagated vegetatively, the phytosanitary status of the propagation material (both the rootstock and the variety) can have a profound effect on the lifetime and health of the new plantations. The fast evolution of sequencing techniques provides a new opportunity for metagenomics-based viral diagnostics. Viral derived small RNAs produced by the host immune system during viral infection can be sequenced by next-generation techniques and analyzed for the presence of viruses, revealing the presence of all known viral pathogens in the sample. This method is based on Illumina sequencing of short RNAs and bioinformatics analysis of virus-derived small RNAs in the host. Here we describe a protocol for this challenging technique step by step with notes, in order to ensure success for every user.

Virus-induced gene silencing (VIGS) uses recombinant viruses to study gene function; however, the effect of the virus vector itself on the gene expression of the host is not always considered. In our work, we investigated non-targeted gene expression changes of the host in order to see how often these changes appear. Effects of various VIGS vector infections were analysed by monitoring gene expression levels of housekeeping genes by Northern blot analysis in four different hosts. We found that non-targeted changes happens very often. More importantly, these non-targeted effects can cause drastic changes in the gene-expression pattern of host genes that are usually used as references in these studies. We have also found that in a tobacco rattle virus (TRV)-based VIGS, the presence of foreign sequences in the cloning site of the vector can also have a non-targeted effect, and even the use of an internal control can lead to unpredicted changes. Our results show that although VIGS is a very powerful technique, the VIGS vector, as a pathogen of the host, can cause unwanted changes in its gene-expression pattern, highlighting the importance of careful selection of both the genes to be tested and those to be used as references in the planned experiments.