Vitiello, Alessia (2018) TRANSCRIPTOME CHARACTERIZATION AND TRANSCRIPTIONAL REPROGRAMMING OF ZUCCHINI PLANT IN RESPONSE TO APHIS GOSSYPII FEEDING. [Tesi di dottorato]

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Zucchini (Cucurbita pepo L.) belongs to the Cucurbitaceae family and ranks among the highest-valued vegetables worldwide. It is widely cultivated in temperate region where one of the main problem related to its cultivation is the damage imposed by the cotton/melon aphid Aphis gossypii (Homoptera: Aphididae). Aphis gossypii is a polyphagous aphid which can both directly and indirectly affects host plant by inducing leaf curling and necrosis and vectoring several plant viruses. Plants can defend themselves against aphids using different strategies. A direct response activates a deep transcriptional reprogramming which leads to de novo synthesis of proteins and molecules implicated in defence, including production of herbivore induced volatile organic compounds (HI-VOCs), which can indirectly attract herbivore natural enemies. In the present study, the mRNA from un-infested (control) and infested leaves by A. gossypii of C. pepo cultivar “San Pasquale” was sequenced to obtain a de novo transcriptome assembly to be used as reference for gene expression profiling. Leaf material was collected from control and infested (10 adult A. gossypii) plants at three different time points (24, 48, 96 hours post infection; hpi) and RNA was extracted. Illumina sequencing generated ~34 million of paired-end reads of 101 nucleotides in length per sample. Short reads were pre-processed and, then, de novo assembled using Velvet/Oases and CAP3 tools into a non-redundant set of 71,648 transcripts. Approximately 94% of the assembled transcripts contains coding sequences that could be translated into proteins, and ~70% of transcripts was successfully annotated using BLAST similarity-based searches and Blast2GO. Furthermore, BLASTn comparisons with the publically available C. pepo transcriptome resulted in 1,313 transcripts exclusively assembled in the aphid-challenged transcriptome. Following transcriptome assembly, a dataset of 42,517 sequences, in which each gene locus was represented only once, was filtered out and used as reference for read mapping and differentially expressed gene (DEG) call. A total of 766 transcripts was differentially expressed (FDR< 0.05; -2˂logFC˃2). At 24 hpi, 158 transcripts were influenced by aphid infestation. The number of affected transcripts increased to 565 at 48 hpi and declined to 179 transcripts at 96 hpi. The analysis of DEGs highlighted the modulation of genes involved in hormone-related defence pathways. Among these, SA-related genes were found mainly up-regulated assuming an important role in “San Pasquale”-A. gossypii interaction. Furthermore, a significant negative effect on aphid fixing behaviour was observed on zucchini plants pre-treated with synthetic methyl salicylate (MeSA). Zucchini plant response was also characterised by the overexpression of genes involved in primary metabolic processes as well as cell wall modification. Interestingly, several aphid-derived transcripts were discovered among zucchini DEGs. Even if no conclusive evidence can be drawn, we hypothesized that these mRNAs might play a role in modulation of plant direct and/or indirect response. Finally, analysis of VOCs emitted by zucchini plants infested with 10 adult aphids, for 48 h, showed a significant reduction in (E)-caryophyllene emission, whereas emission levels of other volatiles were not affected. Conversely, a significant increase in (E)-caryophyllene emission was observed when plants were infested with 300 adult aphids, for 96 h. Moreover, olfactometer bioassays revealed that synthetic (E)-caryophyllene was attractive to female Aphidius colemani parasitic waps, widely used as biological control agents for A. gossypii. Taken together, these results suggest (E)-caryophyllene may play an important role in zucchini plant indirect defence responses. Our study allows to elucidate, for the first time, the molecular mechanisms activated by zucchini plants in response to A. gossypii infestation during a compatible interaction.

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