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Searching for the Mechanism That Mediates the Mefenoxam-Acquired Resistance Phenomenon in Phytophthora Infestans and How It Is Regulated

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Searching for the Mechanism That Mediates the Mefenoxam-Acquired Resistance Phenomenon in Phytophthora Infestans and How It Is Regulated Searching for the mechanism that mediates the mefenoxam-acquired resistance phenomenon in Phytophthora infestans and how it is regulated Juliana González Tobón Director: Giovanna Danies Turano Co-director: Silvia Restrepo Restrepo Department of Biological Sciences Faculty of Sciences Universidad de los Andes October 21st 2018 1 1 Searching for the mechanism that mediates the mefenoxam-acquired resistance 2 phenomenon in Phytophthora infestans and how it is regulated 3 4 Juliana González-Tobón1, Richard Childers2, Alejandra Rodríguez1, William Fry3, Kevin L. 5 Myers3, Keith L. Perry3, Jeremy R. Thompson3, Silvia Restrepo1, and Giovanna Danies4,* 6 7 1 Department of Biological Sciences, Universidad de los Andes, Bogotá, Colombia 8 2 Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, 9 USA 10 3 School of Integrative Plant Science, Plant Pathology and Plant-Microbe Biology Section, 11 Cornell University, Ithaca, NY, USA 12 4 Department of Design, Universidad de los Andes, Bogotá, Colombia 13 14 *Corresponding author: G. Danies; E-mail address: [email protected] 15 2 16 Abstract 17 Phytophthora infestans, the causal agent of late blight disease of potatoes and tomatoes, is 18 mainly controlled by the use of fungicides. Isolates of P. infestans that are resistant to commonly 19 used fungicides have already been reported. Aside from natural resistance, several studies have 20 shown that isolates of P. infestans that are originally sensitive to the fungicide mefenoxam are 21 able to acquire resistance to this fungicide when exposed to sub-lethal concentrations of it. This 22 phenomenon termed ‘mefenoxam-acquired resistance’ has been observed in different species of 23 the Phytophthora genus and seems to be unique to mefenoxam. The first objective of this study 24 was to elucidate the molecular mechanism mediating this acquisition of resistance. For this 25 purpose, we were specifically interested in determining the genes that might be associated with 26 the acquisition of resistance to mefenoxam in Phytophthora infestans. Additionally, we wanted 27 to investigate if the RNA polymerase I (pol I) could be involved in its occurrence. Our results 28 indicate no clear interaction between RNA pol I and the acquisition of resistance in P. infestans. 29 However, differentially expressed genes that might be related to this phenomenon were 30 determined and classified in seven functional groups. Altogether they seem to be involved in a 31 pleiotropic drug resistance (PDR) phenotype, thus elucidating a plausible mechanism for this 32 special type of resistance to mefenoxam. The second objective was to investigate if there is a 33 regulatory epigenetic process, such as small non-coding RNAs, adenine DNA methylation or 34 chromatin remodeling, mediating this mechanism. Interestingly, several small ncRNAs were 35 found to be differentially expressed in the originally sensitive isolates when comparing them 36 before and after acquiring resistance. The majority of these small ncRNAs seem to be 37 specifically related to some of the genes found to be mediating the PDR phenotype, thus 38 accounting for a possible regulatory system to this phenomenon. 3 39 Introduction 40 Phytophthora infestans, a plant pathogenic oomycete, is the causal agent of late blight disease of 41 potatoes and tomatoes. This disease was responsible for the great Irish famine in the mid XIX 42 century and it still causes billion dollar losses worldwide (CIP, 2004). The principal control 43 strategy for this disease, is the use of fungicides (Schoina & Govers, 2015). However, 44 individuals of P. infestans can present natural resistance against them (Fry et al., 2013). 45 Moreover, it has been shown that isolates of P. infestans are able to acquire resistance in vitro to 46 one of the most commonly used fungicides, mefenoxam (containing primarily the active R- 47 enantiomer of metalaxyl) (Childers et al., 2015; Monkiedje & Spiteller, 2002). 48 The phenomenon of mefenoxam-acquired resistance was originally reported in studies 49 dating back to 1979 and 1981 (Bruin & Edgington, 1981; Staub, Dahmen, Urech, & Schwinn, 50 1979). Childers et al., (2015) showed that the acquisition of mefenoxam resistance occurred on 51 different genotypes of P. infestans after a single passage on mefenoxam-containing medium 52 amended with sub-lethal concentrations of the fungicide. Furthermore, they showed that after a 53 few passages on mefenoxam-free medium the isolates that had acquired resistance slightly 54 recovered their sensitivity. Recently, in González-Tobón et al., (in preparation) it was shown that 55 the phenomenon also occurs in isolates of a newly described species termed Phytophthora 56 betacei, closely related to P. infestans (Mideros et al., 2018). Interestingly, in this same study it 57 was shown that the acquisition of resistance to fluopicolide and cymoxanil does not seem to 58 occur. These fungicides contain different chemical compounds (benzamide and cyanoacetamide- 59 oxime, respectively) to that of mefenoxam (phenylamide) (FRAC, 2017). 60 The molecular mechanism behind the phenomenon of mefenoxam-acquired resistance is 61 still unknown. Childers et al., (2015) implemented a whole-transcriptome sequencing assay, to 4 62 isolates of P. infestans before and after the acquisition of resistance, and found a set of genes that 63 were most highly differentially expressed between these two conditions. Mainly, two ABC 64 transporter proteins, the phospholipase D (PLD) gene, some RXLR effectors and several 65 conserved hypothetical proteins were identified. These proteins have been reported in organisms 66 from different phylogenetic lineages but have not been functionally characterized (Galperin & 67 Koonin, 2004). From the 32 genes that were found to be differentially expressed and common 68 among the four isolates tested, almost half of them were annotated as conserved hypothetical 69 proteins. From these, seven had a blastp hit that provided information about it. However, all the 70 others remained unknown (Childers et al., 2015). 71 On the other hand, given that mefenoxam is a phenylamide fungicide known to inhibit 72 the synthesis of rRNAs (Leen C. Davidse, Gerritsma, Ideler, Pie, & Velthuis, 1988; Fisher & 73 Hayes, 1982). a modification or alteration on this process might be expected. So far, it has been 74 reported that metalaxyl has a negative effect on the incorporation of uridine into RNA (L. C. 75 Davidse, 1990). Also, it is known that only a part of the synthesis of rRNAs is sensitive to the 76 fungicide because only RNA Polymerase I (RNA Pol I) is affected by it when in complex with 77 its template (L. C. Davidse, 1990). However, additional information on this matter is not 78 available. 79 A widely known mechanism used by bacteria to respond towards changing or extreme 80 environmental conditions is the use of alternate sigma factors, the recognizing elements of the 81 bacterial RNA polymerase (Decker & Hinton, 2013). These alternate factors seem to be quite 82 important for bacterial pathogenesis (Decker & Hinton, 2013) as seen, for example, in 83 Pseudomonas aeruginosa (Ishimoto & Lory, 1989) and Clostridium difficile (Mani & Dupuy, 84 2001). A similar situation has been reported for RNA Polymerase II in eukaryotes, where this 5 85 enzyme can alternate among a set of binding sequences and transcription factors in order to use 86 available promoters efficiently (Decker & Hinton, 2013). Even though this has not been yet 87 reported for the RNA Polymerase I, a similar mechanism might be expected (Decker & Hinton, 88 2013). Specially under situations that force the organisms to overcome a certain pressure. 89 It was previously suggested in Childers et al., (2015) that a mutation seemed unlikely 90 because of the speed on which the acquisition of mefenoxam resistance occurs, its apparent 91 reversibility, and its occurrence in different genotypes of P. infestans. Therefore, a regulatory 92 mechanism mediating the molecular process behind it is most plausible. An epigenetic 93 mechanism has been considered (Childers et al., 2015). The four principal epigenetic processes, 94 chromatin remodeling, small non-coding RNA regulation (ncRNA), histone tail modifications 95 such as methylation and acetylation, and m6A-type DNA methylation (Chen et al., 2017; Kasuga 96 & Gijzen, 2013; Raffaele et al., 2010; Vetukuri et al., 2011) have been reported in P. infestans. 97 This study aimed to investigate both the mechanism behind the phenomenon of 98 mefenoxam-acquired resistance in P. infestans as well as the regulatory process mediating it. For 99 our first objective, we were specifically interested in determining the genes that might be 100 associated with the acquisition of resistance to mefenoxam in P. infestans. Additionally, we 101 wanted to investigate if the RNA Pol I could be involved in its occurrence. Mainly, by 102 evidencing changes on the 25S and 18S rRNAs genes’ expression or by means of alternative 103 enzyme subunits encoded in the P. infestans genome. For our second objective, we investigated 104 if there is a regulatory epigenetic mechanism, such as small ncRNAs, adenine DNA methylation, 105 or histone modifications, mediating the molecular process that accounts for the mefenoxam- 106 acquired resistance phenomenon. 107 6 108 Materials and Methods 109 Isolates and growth conditions. 110 Three isolates of P. infestans were evaluated in this study, US140120 (US-23 clonal lineage), 111 IMK-1 (US-22 clonal lineage), and RC1#10 (EC-1 clonal lineage). Isolates US140120 and IMK- 112 1 are naturally sensitive to mefenoxam and have been shown to acquire resistance to mefenoxam 113 after exposure to sub-lethal concentrations of the fungicide (Childers et al., 2015); González- 114 Tobón et al., in preparation). Isolate RC1#10 is naturally resistant to mefenoxam. All isolates 115 were kept and routinely transferred onto pea agar (120 g of frozen pea, 15 g of agar, 20 g of 116 sugar, and 2 g of CaCO3 for 1 L of medium) (Jaime-Garcia et al., 2000) and incubated at 20 ± 0.5 117 °C. 118 119 Initial sensitivity, acquisition of resistance to mefenoxam, and total RNA extraction.
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