Reference genes validation and cutinase relative expression in response to heat shock on the fungus Lasiodiplodia theobromae

PAOLINELLI MARCOS; GALINDO-SÁNCHEZ CLARA ELIZABETH; HERNÁNDEZ-MARTÍNEZ RUFINA

Bozeman

Congreso; APS Pacific Division Meeting; 2014

L. theobromae is a phytopathogenic fungus, reported as the most aggressivepathogen on grapevine among the Botryosphaeriaceae, and with higherincidence in warmer regions. Transcriptomic response of some fungi exposedto heat-stress is similar to the observed during plant infection, suggesting thatpathogenic factors are induced with high temperatures. The aim of this workwas to study the in vitro expression of the pathogenic factor cutinase inresponse to heat shock. To achieve this, total RNA was isolated from thefungal mycelium collected at 10 or 120 min after heat shock (1 h at 42°C)grown in Vogel?s salts with or without 1% grapevine wood (GW). Controlsnon-exposed to heat stress, and two biological replicates per condition wereconsidered. qRT-PCR was done to amplify gene fragments of actin (Act),glyceraldehyde-3-phosphate dehydrogenase (Gapdh), b-tubulin (Tub),elongation factor-1a (Ef), and cutinase (Cut). The quantification cycleobtained for each target/sample combination was used to evaluate the stabilityof the reference genes Act, Gapdh, Tub and Ef, through the GeNorm,NormFinder and Bestkeeper applications. The pair Tub/Ef was the beststrategy to normalize the cutinase expression in all the conditions evaluated.The cutinase was overexpressed at 120 min after heat-shock, when the funguswas grown in the presence of GW but not in its absence. Cutinase degradescuticle, the main barrier that covers the aerial part of the plants. Although ithas been reported that L. theobromae infection occurs mainly through pruningwounds, the capacity to breakdown the cuticle is highlighted by the cutinaseexpression in the evaluated conditions, suggesting that adhesion