Resistance-breaking strains of tomato spotted wilt virus hamper photosynthesis and protein synthesis pathways in a virus accumulation-dependent manner in Sw5-carrying tomatoes

Tomato spotted wilt virus (TSWV; Orthotospovirus tomatomaculae) is one of the major agricultural threats due to its worldwide distribution and broad host range, including many important crops. In Italy, TSWV has a significant economic impact on tomato (Solanum lycopersicum L.) in the field. In the last decades, several tomato varieties carrying the Sw-5b gene for the resistance to TSWV have been released. In the virus-host evolution history, the wide use of the Sw-5b gene in tomato varieties was counteracted by the emergence of the so-called Sw5-Resistance-Breaking (SRB) strains, which overcome the resistance. We investigated the interaction between Sw5b-carrying tomatoes and SRB TSWV to elucidate the molecular mechanisms underlying the resistance breakage. Transcriptome sequencing (RNA-Seq) was used to analyze 18 samples (leaves) collected from field-grown tomatoes naturally infected by TSWV in Italy. The samples were divided into four groups based on TSWV symptoms and titer: ASY (asymptomatic leaves with an average of 0.7×102 TSWV reads per million, RPM) and three symptomatic samples, i.e., SYM1 (3.3×104 RPM), SYM2 (7×104 RPM), and SYM3 (1.7×105 RPM). An increase in virus titer resulted in a higher number of differentially expressed genes (DEGs), which ranged from 33 to 44% of the whole transcriptome when SYM1 or SYM3 were compared to ASY, respectively. Photosynthesis, protein biosynthesis and translocation were the main down-regulated biological processes, while enzyme families such as oxidoreductases and transferases, genes related to the response to biotic stimuli, solute transport, and vesicle trafficking were overall up-regulated. Remarkably, the expression of ca. 48% of genes of the whole transcriptome was significantly (P<0.05) correlated (positively or negatively) to the virus titer, and in 6% of cases (over 2000 genes) the correlation was very high (i.e., absolute value of R2>0.85). This finding was verified by a quantitative reverse transcription PCR assay in a greenhouse experiment with a Sw5b-tomato variety artificially inoculated with an SRB TSWV strain. The analysis of 15 genes in 12 samples with a TSWV titer ranging from 0 to 3.2×105-fold change confirmed the expected trend (positive or negative) of the gene expression × virus titer correlation for all the genes, with high statistical significance for seven of them. In conclusion, we elucidated the transcriptional changes induced by SRB TSWV on Sw5b-carrying tomatoes and showed that they are proportional to the TSWV titer. A number of genes can be considered as good markers of TSWV infection. Our finding highlights the importance of routinely determining the virus titer in plant-virus interaction studies to better interpret the results.