TRANSCRIPTOMIC AND ELECTRON-MICROSCOPY INVESTIGATIONS ON GRAPEVINE YELLOW MOSAIC SYMPTOMS ASSOCIATED WITH GRAPEVINE FANLEAF VIRUS

Yellow mosaic (YM) of grapevine caused by grapevine fanleaf virus (GFLV, Nepovirus, transmitted by root nematodes) consists of a persistent, yellow chrome discoloration occurring on the whole blade of the leaves, during spring and summer. Generally, the symptom reverts to a normal green color by late summer and is often observed in several vine branches, while others on the same plant show normal pigmentation. It could be accompanied or not by the most typical leaf malformation and degeneration caused by the same agent, which systemically infects grapevines. While different cultivars respond with the same phenotype recurrently over the years, serological and molecular diagnosis cannot discriminate different GFLV isolates or variants. To shed light on the molecular mechanism of the host plant-virus interaction leading to this syndrome, three YM-affected, field-grown vines of a local white wine cultivar were selected in Apulia vineyards (Southern Italy) and sampled in both green and yellow leaf tissues in July 2019. Total RNAs from these six samples were polyA enriched and prepared for RNA-seq (Illumina^®, 150 paired-ends). Concurrently, small RNA fractions from the same samples were also sequenced. Quality-checked reads were de novo assembled in contigs by rnaSPAdes, then annotated using BLASTN/X vs nt and nr databases, respectively, thus allowing either the identification of viruses and viroids or the annotation of the de novo assembled grapevine transcriptome. Mapping of reads, differential gene and miRNA expression were performed by bowtie2 and DESeq2, respectively. The de novo assembled transcriptome was used as a reference for miRNA/siRNA target prediction by TAPIR.

The virome analysis detected the presence of virus and viroid species commonly affecting grapevine, without showing a discrimination pattern between YM and green leaf tissues. GFLV was detectable showing a higher genome coverage than the other viruses. However, RT-qPCR of GFLV demonstrated that its titre was similar in both leaf tissues, while sequence comparison indicated that the genetic diversity was correlated with the source plant accession, not with the symptom. More than 2100 differentially expressed (DE) genes were identified. A general picture of oxidative stress was observed as prevalent in yellow leaf tissues (overexpression of mitochondrial ubiquitin oxidase and indole-pyruvate monooxygenase), with an altered expression of the photosynthetic pathway (photosystem II reaction center protein, chlorophyll a-b binding protein 40, Mg-protoporphyrin cyclase). Significant overexpression of genes related to flavonoid and anthocyanin metabolism was depicted in the green leaf tissues. About 30 transcripts were selected for further validation by RT-qPCR. Genes showing the highest fold-change values in both leaf tissues were also used in a monthly analysis, from May to September, conducted on the same vines in the season 2022. Expression values for the July sampling were perfectly aligned with the RNA-seq DE data.

DE miRNAs were also consistently validated. MiR403, overexpressed in yellow leaf tissues, probably targets AGO2, thus affecting the silencing pathway. MiR399 and miR3633, overexpressed in the green leaf tissues, are also putatively involved in mRNA targeting and their validation by 5' RACE is ongoing. Finally, a noteworthy result is the chloroplast analysis in EM. In YM leaf tissues, chloroplasts did not evolve from proplastids and were significantly vesiculated and altered. Indeed, two relevant genes involved in chloroplast maturation, chloroplast unusual positioning protein and curvature thylakoid 1 protein were heavily downregulated in YM leaf tissues. The hypothesis that summer high temperatures can influence the symptom reversion to normal green deserves to be explored in further studies.