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Genome-wide miRNA and piRNA profiles of the vector whitefly, Bemisia tabaci during feeding on Tomato yellow leaf curl virus-infected tomato

ABSTRACT: The whitefly, Bemisia tabaci MEAM1 is a devastating vector capable of transmitting hundreds of plant viruses, including Tomato yellow leaf curl virus (TYLCV), to important food and fiber crops. Here we performed genome-wide profiling of micro RNAs (miRNAs) and piwi-interacting RNAs (piRNAs) in whiteflies after feeding on TYLCV-infected tomato or uninfected tomato for 24, 48 and 72 h. Overall, 160 miRNAs were discovered, 68 of which were conserved and 92 were B. tabaci-specific miRNAs. Majority of the genes that were predicted to be targeted by miRNAs had gene ontologies related to metabolic processes. We identified two miRNAs that were differentially expressed in whiteflies when fed on TYLCV-infected tomato compared to whiteflies that fed on uninfected tomato. The identified piRNAs were expressed as clusters throughout the whitefly genome. A total of 53 piRNA clusters were expressed across all time points and treatments, while 5 piRNA clusters were exclusively expressed in whiteflies that fed on TYLCV-infected tomato, and 24 clusters were exclusively expressed in whiteflies that fed on uninfected tomato. Approximately 62% of all identified piRNAs were derived from non-coding sequences that included intergenic regions, introns, and UTRs with unknown functions. The remaining 38% of piRNAs were derived from coding sequences (CDS) and repeat elements. Transposable elements targeted by piRNA clusters included both class I retrotransposons such as Gypsy, Copia, and LINEs and class II DNA transposons such as MITE, hAT, and TcMar. Lastly, six protein coding genes were targeted in whiteflies that fed on TYLCV-infected tomato. Information on how TYLCV influences miRNA and piRNA expression in whiteflies provides a greater understanding of regulatory pathways involved in mediating whitefly-virus interactions, and will facilitate the identification of novel targets for RNAi control.

ORGANISM(S): Bemisia tabaci

PROVIDER: GSE111343 | GEO | 2019/02/28

REPOSITORIES: GEO

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Project description:Tomato seeds (S. lycopersicum ‘Fl Lanai’) were germinated under greenhouse conditions maintained at 24°C-29°C in flat trays (BWI Apopka, Catalog Number GPPF72S7X) filled with Sungro Horticulture soil (Metro-mix 830, BWI Apopka, Cat# TX830). Two weeks post emergence seedlings were transplanted to 4” pots using the same soil and transferred to a Conviron walk-in growth chamber (CMP6060) for the remainder of the experiment. Conviron conditions include a 14h/10h light/dark cycle maintained at 28°C, and plants were fertilized weekly (20-20-20). To prevent cross contamination, tomato plants were confined to insect proof cages at all times (BioQuip 1450NS68). Four weeks after transplanting, 40 whiteflies (B. tabaci MEAM1) were collected from virus free or Tomato Mottle Virus (ToMoV) established colonies via aspiration and moved into a clip cage placed on the 4th true leaf of each tomato plant as previously described38. Whiteflies were reared cabbage (Brassica oleracea), while viruliferous whiteflies were reared on ToMoV infected tomato from colonies established in the Polston lab. For all plants in this study, feeding was halted after 3 days of whitefly feeding (3 DPI) by gentle removal of clip cages and whitefly termination using insecticidal soap (Garden Safe, 1% of potassium salts of fatty acids). For the samples referred to as “local”, the tomato leaf bound within the clip cages was immediately removed and snap frozen for protein extraction. For the samples designated “systemic”, the plants were allowed to continue growing for 7 additional days after clip cage removal and whitefly termination, at which point the 9th leaf was excised and snap frozen. Plants used for collection of local leaves at 3 DPI were not used for the collection of systemic leaves 10 DPI. For both local and systemic leaves collected, we also included a no treatment control (NTC) that was subjected identically to clip cage and insecticidal soap applications, but without the addition of whitefly or ToMoV. Our experiment therefore consists of a no-treatment control (NTC), a whitefly treatment (+WF), and a viruliferous whitefly (+WFV) treatment for both local (4th true leaf, 3 DPI) and systemic leaves (9th true leaf, 10 DPI). The presence of ToMoV in all infected plants was confirmed via Nanopore sequencing. Briefly, Tomato genomic DNA was extracted from five systemic leaf samples using the PureGene tissue DNA isolation kit (product # 158667; QIAGEN, Valencia, CA, USA), following the manufacturer’s protocol and stored at -80°C until needed. Library preparation was performed using the Rapid Sequencing Kit RBK004 protocol (Oxford Nanopore Technologies) and loaded onto a 9.4.1 flow cell in a MinION connected to a MinIT with live base calling enabled. Resulting sequencing reads for each sample were mapped to both ToMoV A component (GenBank accession: L14460) and ToMoV B component (GenBank accession: L14461) sequences.

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Genome-wide miRNA and piRNA profiles of the vector whitefly, Bemisia tabaci during feeding on Tomato yellow leaf curl virus-infected tomato

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