Adaptive genomic structural variation in the grape powdery mildew pathogen, Erysiphe necator

BMC Genomics Laura Jones 1 Summaira Riaz 1 Abraham Morales-Cruz 1 Katherine CH Amrine 1 Brianna McGuire 0 W Douglas Gubler 0 M Andrew Walker 1 Dario Cantu 1 0 Department of Plant Pathology, University of California Davis , One Shields Ave, Davis, CA 95616 , USA 1 Department of Viticulture and Enology, University of California Davis , One Shields Ave, Davis, CA 95616 , USA Jones et al. - Adaptive genomic structural variation in the grape powdery mildew pathogen, Erysiphe necator Open Access Adaptive genomic structural variation in the grape powdery mildew pathogen, Erysiphe necator Background: Powdery mildew, caused by the obligate biotrophic fungus Erysiphe necator, is an economically important disease of grapevines worldwide. Large quantities of fungicides are used for its control, accelerating the incidence of fungicide-resistance. Copy number variations (CNVs) are unbalanced changes in the structure of the genome that have been associated with complex traits. In addition to providing the first description of the large and highly repetitive genome of E. necator, this study describes the impact of genomic structural variation on fungicide resistance in Erysiphe necator. Results: A shotgun approach was applied to sequence and assemble the genome of five E. necator isolates, and RNA-seq and comparative genomics were used to predict and annotate protein-coding genes. Our results show that the E. necator genome is exceptionally large and repetitive and suggest that transposable elements are responsible for genome expansion. Frequent structural variations were found between isolates and included copy number variation in EnCYP51, the target of the commonly used sterol demethylase inhibitor (DMI) fungicides. A panel of 89 additional E. necator isolates collected from diverse vineyard sites was screened for copy number variation in the EnCYP51 gene and for presence/absence of a point mutation (Y136F) known to result in higher fungicide tolerance. We show that an increase in EnCYP51 copy number is significantly more likely to be detected in isolates collected from fungicide-treated vineyards. Increased EnCYP51 copy numbers were detected with the Y136F allele, suggesting that an increase in copy number becomes advantageous only after the fungicide-tolerant allele is acquired. We also show that EnCYP51 copy number influences expression in a gene-dose dependent manner and correlates with fungal growth in the presence of a DMI fungicide. Conclusions: Taken together our results show that CNV can be adaptive in the development of resistance to fungicides by providing increasing quantitative protection in a gene-dosage dependent manner. The results of this work not only demonstrate the effectiveness of using genomics to dissect complex traits in organisms with very limited molecular information, but also may have broader implications for understanding genomic dynamics in response to strong selective pressure in other pathogens with similar genome architectures. * Correspondence: 1Department of Viticulture and Enology, University of California Davis, One Shields Ave, Davis, CA 95616, USA Full list of author information is available at the end of the article Background Grapevine powdery mildew is one of the most widespread and devastating diseases of wine, table and raisin grapes, the vast majority of which are cultivars of Vitis vinifera. This disease is caused by the fungus Erysiphe necator Schw. [syn. Uncinula necator (Schw.) Burr.], an obligate biotroph that can infect all green tissues of a grapevine (Figure 1A-C [1]). Infected leaves exhibit reduced photosynthesis and often undergo premature senescence and abscission. Early berry infection causes berries to crack, and the overall impact on the crop includes decreased yields, increased acidity, and decreased anthocyanin and sugar content of mature fruit [2]. Even low levels of powdery mildew infection on the berries can lead to ruined table grapes and wines with negative sensory attributes and decreased varietal character [2,3]. Most cultivated varieties are susceptible to powdery mildew, and as a consequence growers are forced to apply fungicides to control the disease, often as frequently as every 710 days when disease pressure is high. It has been estimated that as much as 20% of the total costs associated with wine grape production in California goes to expenses related to powdery mildew control [7]. Elemental sulfur was the first effective fungicide recommended for vineyards in 1848 to control powdery mildew, and it continues to be widely used, mainly due to its efficacy and low cost [8]. Although its multi-site mode of action remains effective at controlling powdery mildew, the limitations to sulfur use include phytotoxicity at higher temperatures, the need for application as a protectant at frequent intervals, potential off-characters in wine, and the risk of unintended environmental consequences [9,10]. Since the 1960s, new classes of fungicides have been developed and introduced, many with single-site modes of action and beneficial properties such as systemic effectiveness and longer times between applications [8]. One important class of single-site fungicides is the sterol demethylase inhibitors (DMI), which includes the azole fungicides. DMIs inhibit fungal Figure 1 Powdery mildew disease symptoms and phylogenetic relation of E. necator with other grapevine fungal pathogens and other powdery mildew pathogens. E. necator infections are initiated when conidia come in contact with a susceptible host and germinate, forming hyphae with multilobed appressoria and penetration pegs. Haustoria are formed within the epidermal cell membrane to maintain the parasitic relationship with the host. Young colonies are macroscopically visible and appear white on the surface of (A) leaves, (B) fruit, and other green tissue. Multiseptate conidophores form along the hyphae perpendicularly to the epidermis, and (C) conidia begin to develop within a few days of the initial infection (white bar = 0.2 mm). (D) Phylogenetic relationship of E. necator with other powdery mildews (gray) and grape fungal pathogens (red). The Neighbor-Joining tree was constructed in MEGA5 [4] using the complete rDNA ITS (ITS1, 5.8 rDNA, ITS2). Multiple publicly available ITS sequences per species were used independently to confirm clustering. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches [5]. Only bootstrap values greater than 60 are shown. The evolutionary distances were computed using the Maximum Composite Likelihood method [6] and are in the units of the number of base substitutions per site. The analysis involved 11 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 372 positions in the final dataset. growth by targeting the cytochrome P450 lanosterol C14-demethylase (CYP51, also known as E (...truncated)