Overexpression of the CYP51A1 Gene and Repeated Elements are Associated with Differential Sensitivity to DMI Fungicides in Venturia inaequalis

Abstract

The involvement of overexpression of the CYP51A1 gene in Venturia inaequalis was investigated for isolates exhibiting differential sensitivity to the triazole demethylation inhibitor (DMI) fungicides myclobutanil and difenoconazole. Relative expression (RE) of the CYP51A1 gene was significantly greater (P < 0.0001) for isolates with resistance to both fungicides (MRDR phenotype) or with resistance to difenoconazole only (MSDR phenotype) compared with isolates that were resistant only to myclobutanil (MRDS phenotype) or sensitive to both fungicides (MSDS phenotype). An average of 9- and 13-fold increases in CYP51A1 RE were observed in isolates resistant to difenoconazole compared with isolates with MRDS and MSDS phenotypes, respectively. Linear regression analysis between isolate relative growth on myclobutanil-amended medium and log10 RE revealed that little to no variability in sensitivity to myclobutanil could be explained by CYP51A1 overexpression (R(2) = 0.078). To investigate CYP51A1 upstream anomalies associated with CYP51A1 overexpression or resistance to difenoconazole, Illumina sequencing was conducted for three isolates with resistance to difenoconazole and one baseline isolate. A repeated element, "EL 3,1,2", with the properties of a transcriptional enhancer was identified two to four times upstream of CYP51A1 in difenoconazole-resistant isolates but was not found in isolates with the MRDS phenotype. These results suggest that different mechanisms may govern resistance to different DMI fungicides in the triazole group.

The origin, versatility and distribution of azole fungicide resistance in the banana black Sigatoka pathogen Pseudocercospora fijiensis

“…More detailed studies would be required in P. fijiensis to decipher whether the insertions we observed corresponds to the movements of a transposon sequence or whether the Pfcyp51 expression might also be regulated by transposons. However, unlike previous reports of promoter insertions with a 199 bp to 5.6 kb-sequence transposon, the promoter insertion in Pfcyp51 is a repeated merely 19 bp fragment, reaching only 100 bp in length, even shorter than insertions in Venturia inaequalis (Schnabel and Jones, 2001;Villani et al, 2016) and Z. tritici (Cools et al, 2012), where transposons were not reported. In other organisms e.g.…”

“…Contrary to our expectations, progeny analyses provided strong evidence that DMI resistance in P. fijiensis is solely based on Pfcyp51 modulation and not on other previously reported mechanisms such as increased efflux (Becher & Wirsel 2012;Leroux & Walker 2011). Despite increasing and accumulating data on fungicide resistance in fugal human and plant pathogens (Chowdhary et al 2013;Eddouzi et al 2013;Guzmán et al 2013;Hollomon 2015;Sun et al 2013;Verweij et al 2013;Villani et al 2016), our study is the first genetic analysis to map the underlying genetic factors for reduced DMI efficacy.…”

“…Strains with reduced sensitivity have usually two, three or more copies of this element. Changes in the promoter region of the cyp51 gene have been described in other fungi, such as truncated derivatives of a LINE-like retrotransposon in Blumeriella jaappi (Ma et al, 2006), a MITElike transposon named PdMLE1 in Penicillium digitatum (Sun et al 2013), a larger transposon of 1.8 kb in A. fumigatus (Albarrag et al, 2011;Verweij et al, 2013) and transcription factors binding site in V. inaequalis (Villani et al, 2016). More detailed studies would be required in P. fijiensis to decipher whether the insertions we observed corresponds to the movements of a transposon sequence or whether the Pfcyp51 expression might also be regulated by transposons.…”

“…Common mechanisms described for the loss of sensitivity to DMIs are increased efflux of the fungicides from the cells Leroux et al 2010), adaptation and overexpression of the fungicide target gene (Bolton et al 2016;Chong et al 2016b;Cools et al 2012;Díaz-Trujillo et al 2016a;Villani et al 2016) and cellular alterations that reduce the toxicity of the fungicides, such as modulation of the targeted biosynthesis pathway (Cowen 2008). Recently, we showed that modulation of the promoter and coding domain of Pfcyp51 explains reduced DMI sensitivity (Chong et al 2016b;Díaz-Trujillo et al 2016a).…”

“…The link between DMI fungicides overuse and the occurrence of reduced efficacy and concurring genetic variation at the target site has been demonstrated in many fungal species (Becher & Wirsel 2012;Villani et al 2016;Warrilow et al 2013). The commonest observed genetic mechanisms of DMI resistance are nonsynonymous point mutations in the coding region of the cyp51 gene resulting in modified versions of the CYP51 protein, and changes in the cyp51 gene promoter resulting in elevated expression levels (Akins & Sobel 2009;Albarrag et al 2011;Albertini et al 2003;Bean et al 2009;Bolton et al 2016;Cools et al 2012;Délye et al 1997;Díaz-Trujillo et al 2016a;Dyer et al 2000;Eddouzi et al 2013;Hamamoto et al 2000;Ma et al 2006;Mellado et al 2007;Schnabel & Jones 2000;Verweij et al 2013).…”