Agricultural production is challenged by considerable losses in crop yield and post-harvest storage due to fungal diseases. Among fungal pathogens Botrytis cinerea is considered the second in the ‘top ten’ for its economic importance due to its broad host range. Traditional fungicides can be ineffective and harmful and with increasing fungicide resistance, new eco-friendly and sustainable technologies are required to protect crops. In this scenario, there is a great interest for RNA interference (RNAi)-based fungicides, that are emerging recently for their potential uses. The exogenous application of double-stranded RNA (dsRNA) triggers gene silencing in a sequence specific manner. In our study, we used the lettuce-B. cinerea (B05.10) pathosystem to assess the effect of topical application of a noncoding dsRNA. We selected as target gene the mitogen‐activated protein kinase (MAPK) Bmp3 (BcBmp3) that plays important roles in pathogenicity/virulence (surface sensing of germ tubes, host penetration and lesion formation) as well as in growth retardation (Rui and Hahn 2007, Mol. Plant Pathol., 8, 173-184). After performing a prediction analysis of small interfering RNAs (siRNAs), a 427 nucleotides long dsRNA (BcBmp3-dsRNA) was selected as the best fitting construct. In vitro fungal growth was delayed using the BcBmp3-dsRNA with a great reduction of 95.6% at 48 h, subsequently restored at 96 h. Anyway, the expression of BcBmp3 was significantly suppressed at 48 and 96 h post-treatment (34.6% and 87.4%, respectively). Detached lettuce leaves were artificially inoculated with a conidial suspension of B. cinerea after being topically treated with the construct. They showed reduced necrotic areas at five days post-inoculation, 89.6% less than the control. This result was confirmed by qRT-PCR analysis where a remarkably reduced level of Bmp3 transcripts of 68.2% was found. Lastly, we analysed the effect of BcBmp3-dsRNA on conidial germination whose percentage was reduced by 52.2% at 6 h compared to the control. In summary, this study highlighted that the in vitro and in vivo application of BcBmp3-dsRNA led to gene knockdown with a delay in conidial germination, an evident growth retardation and a strongly reduction of the necrotic lesions on detached lettuce leaves. These promising results are interesting for the development of an RNAi-based fungicide against B. cinerea. SIGS experiments with naked dsRNA or dsRNA loaded onto nanoparticles are in progress.

Bmp3 gene knockdown by topical application of dsRNA affects Botrytis cinerea growth and virulence on Lactuca sativa

Maria Spada
;
Claudio Pugliesi;Marco Fambrini;Susanna Pecchia
2021-01-01

Abstract

Agricultural production is challenged by considerable losses in crop yield and post-harvest storage due to fungal diseases. Among fungal pathogens Botrytis cinerea is considered the second in the ‘top ten’ for its economic importance due to its broad host range. Traditional fungicides can be ineffective and harmful and with increasing fungicide resistance, new eco-friendly and sustainable technologies are required to protect crops. In this scenario, there is a great interest for RNA interference (RNAi)-based fungicides, that are emerging recently for their potential uses. The exogenous application of double-stranded RNA (dsRNA) triggers gene silencing in a sequence specific manner. In our study, we used the lettuce-B. cinerea (B05.10) pathosystem to assess the effect of topical application of a noncoding dsRNA. We selected as target gene the mitogen‐activated protein kinase (MAPK) Bmp3 (BcBmp3) that plays important roles in pathogenicity/virulence (surface sensing of germ tubes, host penetration and lesion formation) as well as in growth retardation (Rui and Hahn 2007, Mol. Plant Pathol., 8, 173-184). After performing a prediction analysis of small interfering RNAs (siRNAs), a 427 nucleotides long dsRNA (BcBmp3-dsRNA) was selected as the best fitting construct. In vitro fungal growth was delayed using the BcBmp3-dsRNA with a great reduction of 95.6% at 48 h, subsequently restored at 96 h. Anyway, the expression of BcBmp3 was significantly suppressed at 48 and 96 h post-treatment (34.6% and 87.4%, respectively). Detached lettuce leaves were artificially inoculated with a conidial suspension of B. cinerea after being topically treated with the construct. They showed reduced necrotic areas at five days post-inoculation, 89.6% less than the control. This result was confirmed by qRT-PCR analysis where a remarkably reduced level of Bmp3 transcripts of 68.2% was found. Lastly, we analysed the effect of BcBmp3-dsRNA on conidial germination whose percentage was reduced by 52.2% at 6 h compared to the control. In summary, this study highlighted that the in vitro and in vivo application of BcBmp3-dsRNA led to gene knockdown with a delay in conidial germination, an evident growth retardation and a strongly reduction of the necrotic lesions on detached lettuce leaves. These promising results are interesting for the development of an RNAi-based fungicide against B. cinerea. SIGS experiments with naked dsRNA or dsRNA loaded onto nanoparticles are in progress.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11568/1132954
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