Time-Course Microarray Analysis Reveals Differences between Transcriptional Changes in Tomato Leaves Triggered by Mild and Severe Variants of Potato Spindle Tuber Viroid.

Viroids are small non-capsidated non-coding RNA replicons that utilize host factors for efficient propagation and spread through the entire plant. They can incite specific disease symptoms in susceptible plants. To better understand viroid-plant interactions, we employed microarray analysis to observe the changes of gene expression in "Rutgers" tomato leaves in response to the mild (M) and severe (S23) variants of potato spindle tuber viroid (PSTVd). The changes were analyzed over a time course of viroid infection development: (i) the pre-symptomatic stage; (ii) early symptoms; (iii) full spectrum of symptoms and (iv) the so-called 'recovery' stage, when stem regrowth was observed in severely affected plants. Gene expression profiles differed depending on stage of infection and variant. In S23-infected plants, the expression of over 3000 genes was affected, while M-infected plants showed 3-fold fewer differentially expressed genes, only 20% of which were specific to the M variant. The differentially expressed genes included many genes related to stress; defense; hormone metabolism and signaling; photosynthesis and chloroplasts; cell wall; RNA regulation, processing and binding; protein metabolism and modification and others. The expression levels of several genes were confirmed by nCounter analysis.

Viability and genetic stability of potato spindle tuber viroid mutants with indels in specific loops of the rod-like secondary structure.

Maintenance of the rod-like structure of potato spindle tuber viroid (PSTVd), which contains over 20 loops and bulges between double-stranded helices, is important for viroid biology. To study tolerance to modifications of the stem-loop structures and PSTVd capacity for mutation repair, we have created 6 mutants carrying 3-4 nucleotides deletions or insertions at three unique restriction sites, EagI, StyI and AvaII. Differences in the infectivity of these in vitro generated PSTVd mutants can result from where the mutations map, as well as from the extent to which the secondary structure of the molecule is affected. Deletion or insertion of 4 nucleotides at the EagI and StyI sites led to loss of infectivity. However, mutants with deletion (PSTVd-Ava-del) or insertion (PSTVd-Ava-in) of 3 nucleotides (221GAC223), at the AvaII site (loop 20) were viable but not genetically stable. In all analyzed plants, reversion to the wild type PSTVd-S23 sequence was observed for the PSTVd-Ava-in mutant a few weeks after agroinfiltration. Analysis of PSTVd-Ava-del progeny allowed the identification of 10 new sequence variants carrying various modifications, some of them having retained the original three nucleotide deletion at the AvaII site. Interestingly, other variants gained three nucleotides in the deletion site but did not revert to the original wild type sequence. The genetic stability of the progeny PSTVd-Ava-del sequence variants was evaluated in tomato leaves (early infection) and in both leaves and roots (late infection), respectively.