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. 2016 Apr 6;6:24103. doi: 10.1038/srep24103

Insights into the adaptive response of the plant-pathogenic oomycete Phytophthora capsici to the fungicide flumorph

Zhili Pang 1, Lei Chen 1,2, Wenjun Mu 1,3, Li Liu 1, Xili Liu 1,a
PMCID: PMC4822174  PMID: 27050922

Abstract

Phytophthora capsici is an important oomycete plant pathogen that causes significant losses worldwide. The carboxylic acid amide fungicide flumorph has shown excellent activity against oomycete plant pathogens. Despite its potential, there remains concern that the sexual reproduction of oomycete pathogens, which results in genetic recombination, could result in the rapid development of resistance to flumorph. The current study utilized an iTRAQ (isobaric tags for relative and absolute quantitation) based method to compare differences between the proteome of the parental P. capsici isolate PCAS1 and its sexual progeny S2-838, which exhibits significant resistance to flumorph. A total of 2396 individual proteins were identified, of these, 181 were considered to be associated with the adaptive response of P. capsici to flumorph. The subsequent bioinformatic analysis revealed that the adaptive response of P. capsici to flumorph was complex and regulated by multiple mechanisms, including utilising carbohydrate from the host environment to compensate for the cell wall stress induced by flumorph, a shift in energy generation, decreased amino acids biosynthesis, and elevated levels of proteins associated with the pathogen’s response to stimulus and transmembrane transport. Moreover, the results of the study provided crucial data that could provide the basis for early monitoring of flumorph resistance in field populations of P. capsici.

Phytophthora capsici was first reported by Leon H. Leonian in 19221, and is currently regarded as one of the 10 most important oomycete pathogens in molecular plant pathology2. This devastating pathogen has a global distribution and can infect more than 45 species of plants including both crops and weed species3,4,5,6, causing crown, root, and fruit rot7,8,9, which lead to significant economic losses every year2,5,10. Although, crop rotation and other management tools contribute to the control of diseases caused by P. capsici, in practice there is a heavy reliance on fungicides11,12. The carboxylic acid amide (CAA) fungicide flumorph, 4-[3-(3,4-dimethoxyphenyl)-3-(4-fluorophenyl)-1-oxo-2-propenyl] morpholine, which was developed by the Shenyang Research Institute of Chemical Industry of China in 199413, has been patented in China (ZL.96115551.5), the United States (US6020332), and Europe (0860438B1). It is currently registered for the control P. capsici, Phytophthora infestans, Pseudoperonospora cubensis, and Plasmopara viticola in China, and remains an effective fungicide to control diseases caused by P. capsici14.

The sexual reproduction of P. capsici15 plays an important role in its disease cycle initiating infection in host plants5,16, while the resulting genetic recombination can contribute to the development of isolates that exhibit complete insensitivity to certain fungicides17. P. capsici is a heterothallic pathogen that produces two mating types, A1 and A2, and it has been shown that the co-occurrence of both mating types in regions of the United States, South Africa, and the northern provinces of China, can facilitate frequent outcrossing and increase the risk of resistance developing18,19,20,21. Previous studies have shown that flumorph resistance in P. capsici is controlled by two dominant genes, which implies that once resistance has developed it could rapidly spread through a population via both sexual and asexual reproduction22.

Proteomics has become a useful tool for studying the biological effects of fungicides. For example, 2-DE has been used to investigate the global response of Saccharomyces cerevisiae in the early stages of exposure to mancozeb23, while MALDI-TOF-MS/MS has been used to study the mode of action of the fungicide JS399-19 in Fusarium graminearum24, and iTRAQ (isobaric tags for relative and absolute quantitation) technology to study the effect of pyrimorph in P. capsici25. The current study adopted a similar approach, using iTRAQ to compare the response of a wild-type parental P. capsici isolate (PCAS1) and its flumorph resistant sexual progeny (S2-838). The proteomics data produced would hopefully provide a greater understanding of the adaptive mechanisms associated with flumorph resistance in P. capsici, as well as highlighting target proteins for not only the early monitoring of flumorph resistance, especially that associated with sexual reproduction, but also for the design of novel fungicides.

Results

Overview of quantitative proteomics analysis

A total of 2396 individual proteins with at least one unique peptide and protein scores >20 were identified from the wild-type (PCAS1) and flumorph-resistant (S2-838) isolates of P. capsici cultured in the presence or absence of flumorph (1.5 μg/ml or 100 μg/ml, respectively) using iTRAQ-LC-MS/MS analysis (identified protein and peptide information, Supplementary Table S1, S2).

Effect of flumorph on protein levels

In total, 189 and 26 proteins were found to be significantly altered in PCAS1 and S2-838, respectively (Supplementary Table S3, S4). Of the 189 proteins detected in the wild-type isolate PCAS1, a total of 80 were up-regulated, and the other 109 down-regulated. In contrast, the flumorph-resistant isolate S2-838 was much less affected with only 21 up-regulated proteins and 5 down-regulated ones.

Identification of candidate proteins for the adaptive response of P. capsici to flumorph

It was found that 181 proteins were associated with the adaptive response of P. capsici to flumorph, with altered levels of abundance in the wild-type isolate PCAS1, but not in the flumorph-resistant isolate S2-838, when comparing the control cultures to those treated with flumorph (Table 1). The subsequent GO analysis categorized these proteins into 14 functional groups according to their biological activity (Fig. 1). The majority of the proteins fell into just two categories metabolic process (83) and cellular process (54). The other proteins fell into 12 categories including developmental process, cellular component biogenesis, cellular component organization, death, pigmentation, localization, response to stimulus, multicellular organismal process, growth, multi/-organism process, establishment of localization, and biological regulation. However, it should be noted that a single protein can be assigned to more than one category. Metabolic pathway enrichment analysis was then performed by matching the proteins with altered abundance to annotated proteins in the KEGG Pathway database. Although it was not possible to classify a large number of the proteins (51), the majority were assigned to a diverse range of metabolic pathways, including amino acid metabolism, carbohydrate metabolism, energy metabolism, lipid metabolism, nucleobase-containing compound metabolism, response to stimulus, transport, and other metabolic pathway (Fig. 2).

Table 1. Candidate proteins identified by iTRAQ analysis for the adaptive response of P. capsici to flumorph.

Accession Description Pathway
348690896 hypothetical protein PHYSODRAFT_553624 Amino acid metabolism
348690401 Hypothetical protein PHYSODRAFT_553293 Amino acid metabolism
348689700 Hypothetical protein PHYSODRAFT_294643 Amino acid metabolism
348687321 Hypothetical protein PHYSODRAFT_471713 Amino acid metabolism
348685637 Hypothetical protein PHYSODRAFT_326460 Amino acid metabolism
348684607 Hypothetical protein PHYSODRAFT_344705 Amino acid metabolism
348684064 Hypothetical protein PHYSODRAFT_485399 Amino acid metabolism
348683248 hypothetical protein PHYSODRAFT_349622 Amino acid metabolism
348683007 hypothetical protein PHYSODRAFT_284659 Amino acid metabolism
348675955 hypothetical protein PHYSODRAFT_354820 Amino acid metabolism
348675840 Hypothetical protein PHYSODRAFT_260724 Amino acid metabolism
348671280 hypothetical protein PHYSODRAFT_520447 Amino acid metabolism
348668898 Hypothetical protein PHYSODRAFT_564655 Amino acid metabolism
348666294 Hypothetical protein PHYSODRAFT_289076 Amino acid metabolism
262111232 Non-selective Cation Channel-2 (NSCC2) Family Amino acid metabolism
262110913 Glycine amidinotransferase Amino acid metabolism
262109966 Protein transporter Sec61 subunit alpha Amino acid metabolism
262108863 argininosuccinate lyase Amino acid metabolism
262104624 Glutamyl-tRNA synthetase Amino acid metabolism
262102276 Cysteine synthase Amino acid metabolism
262102113 Conserved hypothetical protein Amino acid metabolism
262102109 Eukaryotic translation initiation factor 3 Amino acid metabolism
262101719 Threonyl-tRNA synthetase Amino acid metabolism
262100869 Glu/Leu/Phe/Val dehydrogenase family Amino acid metabolism
262100443 glutathione S-transferase, putative Amino acid metabolism
262100355 5-methlytetrahydropteroyltriglutamate-homocysteine methyltransferease Amino acid metabolism
262097641 Conserved hypothetical protein Amino acid metabolism
262097548 60S ribosomal protein L19-1 Amino acid metabolism
348690023 Hypothetical protein PHYSODRAFT_284519 Carbohydrate metabolism
348688731 Hypothetical protein PHYSODRAFT_284295 Carbohydrate metabolism
348688629 hypothetical protein PHYSODRAFT_343844 Carbohydrate metabolism
348687786 Hypothetical protein PHYSODRAFT_261542 Carbohydrate metabolism
348687768 Hypothetical protein PHYSODRAFT_293395 Carbohydrate metabolism
348687704 hypothetical protein PHYSODRAFT_554034 Carbohydrate metabolism
348686055 Hypothetical protein PHYSODRAFT_354100 Carbohydrate metabolism
348684537 Hypothetical protein PHYSODRAFT_344687 Carbohydrate metabolism
348683824 Hypothetical protein PHYSODRAFT_482943 Carbohydrate metabolism
348683217 Hypothetical protein PHYSODRAFT_358973 Carbohydrate metabolism
348681440 Hypothetical protein PHYSODRAFT_285579 Carbohydrate metabolism
348679829 putative lectin [Phytophthora sojae] Carbohydrate metabolism
348677650 Hypothetical protein PHYSODRAFT_264166 Carbohydrate metabolism
348676929 Hypothetical protein PHYSODRAFT_559636 Carbohydrate metabolism
348675829 Hypothetical protein PHYSODRAFT_302104 Carbohydrate metabolism
348675658 Hypothetical protein PHYSODRAFT_286325 Carbohydrate metabolism
348674156 Putative exo-1,3-beta-glucanase Carbohydrate metabolism
348672383 Hypothetical protein PHYSODRAFT_286936 Carbohydrate metabolism
348670337 Hypothetical protein PHYSODRAFT_564447 Carbohydrate metabolism
348670028 Putative carboxylase Carbohydrate metabolism
348669512 Phosphoglycerate kinase Carbohydrate metabolism
348667991 Hypothetical protein PHYSODRAFT_526336 Carbohydrate metabolism
348667785 Hypothetical protein PHYSODRAFT_565503 Carbohydrate metabolism
348667135 Hypothetical protein PHYSODRAFT_530509 Carbohydrate metabolism
348666811 Hypothetical protein PHYSODRAFT_565653 Carbohydrate metabolism
348666456 Hypothetical protein PHYSODRAFT_341553 Carbohydrate metabolism
348664576 Hypothetical protein PHYSODRAFT_343277 Carbohydrate metabolism
332985070 Enolase Carbohydrate metabolism
262112524 Glucokinase, putative Carbohydrate metabolism
262111992 Fumarate hydratase Carbohydrate metabolism
262111868 Pyruvate carboxylase Carbohydrate metabolism
262111867 Pyruvate carboxylase Carbohydrate metabolism
262111277 D-lactate dehydrogenase Carbohydrate metabolism
262109936 Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit Carbohydrate metabolism
262109887 Succinate dehydrogenase flavoprotein subunit Carbohydrate metabolism
262109798 Acetate kinase Carbohydrate metabolism
262108121 Pyruvate, phosphate dikinase Carbohydrate metabolism
262107807 Lectin, putative Carbohydrate metabolism
262104765 Phosphate acetyltransferase Carbohydrate metabolism
262103650 Fructose 1,6 bisphosphatase Carbohydrate metabolism
262103560 glyceraldehyde-3-phosphate dehydrogenase Carbohydrate metabolism
262102812 Malate dehydrogenase Carbohydrate metabolism
262101165 lectin, putative [Phytophthora infestans T30-4] Carbohydrate metabolism
262099080 Aldehyde dehydrogenase Carbohydrate metabolism
262098611 Glucan 1,3-beta-glucosidase Carbohydrate metabolism
262098605 Phosphoenolpyruvate carboxykinase Carbohydrate metabolism
262097763 Glucan 1,3-beta-glucosidase Carbohydrate metabolism
262097378 Phosphoglycerate kinase Carbohydrate metabolism
262097374 Pyruvate kinase Carbohydrate metabolism
348689136 Hypothetical protein PHYSODRAFT_552537 Energy metabolism
348683826 Pyrophosphatase Energy metabolism
348679595 Proton pump, proton transport Energy metabolism
348675725 Hypothetical protein PHYSODRAFT_346620 Energy metabolism
348673684 Hypothetical protein PHYSODRAFT_562177 Energy metabolism
348671348 Hypothetical protein PHYSODRAFT_287246 Energy metabolism
262109621 Sulfite reductase [NADPH] subunit beta Energy metabolism
262100029 Plasma membrane H + -ATPase Energy metabolism
262098159 12-oxophytodienoate reductase, putative Energy metabolism
262095198 S-formylglutathione hydrolase Energy metabolism
254576457 NADH dehydrogenase subunit I Energy metabolism
348690480 Hypothetical protein PHYSODRAFT_553352 Lipid metabolism
348679431 Hypothetical protein PHYSODRAFT_557078 Lipid metabolism
348678066 Putative glycosyl hydrolase family 30 protein Lipid metabolism
348677854 Hypothetical protein PHYSODRAFT_285961 Lipid metabolism
262108963 Glucosylceramidase Lipid metabolism
262105919 3-ketodihydrosphingosine reductase Lipid metabolism
262105742 Acyl-CoA dehydrogenase Lipid metabolism
348688828 Hypothetical protein PHYSODRAFT_353568 Nucleobase-containing compound metabolism
348688657 hypothetical protein PHYSODRAFT_294028 Nucleobase-containing compound metabolis
348687452 Hypothetical protein PHYSODRAFT_284079 Nucleobase-containing compound metabolism
348684415 Hypothetical protein PHYSODRAFT_284882 Nucleobase-containing compound metabolism
348677381 Hypothetical protein PHYSODRAFT_354553 Nucleobase-containing compound metabolism
348677150 hypothetical protein PHYSODRAFT_503916 Nucleobase-containing compound metabolism
348673952 Hypothetical protein PHYSODRAFT_286691 Nucleobase-containing compound metabolism
348672301 hypothetical protein PHYSODRAFT_547952 Nucleobase-containing compound metabolism
348671618 Hypothetical protein PHYSODRAFT_435859 Nucleobase-containing compound metabolism
348670008 hypothetical protein PHYSODRAFT_347790 Nucleobase-containing compound metabolism
262107481 Pre-mRNA-splicing factor SF2 Nucleobase-containing compound metabolism
262106006 60S ribosomal protein L15-1 Nucleobase-containing compound metabolism
262104367 NADH-ubiquinone oxidoreductase, putative Nucleobase-containing compound metabolism
262099101 Pre-mRNA-processing-splicing factor 8 Nucleobase-containing compound metabolism
262095673 hypothetical protein PITG_19772 Nucleobase-containing compound metabolism
348684155 hypothetical protein PHYSODRAFT_478148 Others
348684075 hypothetical protein PHYSODRAFT_349787 Others
348683892 hypothetical protein PHYSODRAFT_353864 Others
348683825 putative dehydratase Others
348681277 hypothetical protein PHYSODRAFT_557322 Others
348673004 hypothetical protein PHYSODRAFT_354913 Others
348673003 hypothetical protein PHYSODRAFT_354912 Others
262105863 aldo/keto reductase family Others
262103226 succinate semialdehyde dehydrogenase Others
262099089 succinate dehydrogenase iron-sulfur protein Others
262098735 alcohol dehydrogenase, putative Others
348690141 hypothetical protein PHYSODRAFT_284543 Response to stimulus
348683864 Hypothetical protein PHYSODRAFT_353859 Response to stimulus
348672012 Elicitin Response to stimulus
262110397 glutaredoxin [Phytophthora infestans T30-4] Response to stimulus
262109962 Alkaline phosphatase Response to stimulus
262106782 Superoxide dismutase 2 Response to stimulus
262101058 Metalloprotease family M17 Response to stimulus
262099848 Conserved hypothetical protein Response to stimulus
348678388 ABC transporter ABCA1 lipid exporter family Transport
348690807 Hypothetical protein PHYSODRAFT_349569 Unclassified
348690475 Hypothetical protein PHYSODRAFT_323696 Unclassified
348689826 Hypothetical protein PHYSODRAFT_252686 Unclassified
348688971 Hypothetical protein PHYSODRAFT_537442 Unclassified
348688710 Hypothetical protein PHYSODRAFT_477401 Unclassified
348688366 Hypothetical protein PHYSODRAFT_353487 Unclassified
348687330 Hypothetical protein PHYSODRAFT_284057 Unclassified
348683932 Hypothetical protein PHYSODRAFT_485543 Unclassified
348683032 Hypothetical protein PHYSODRAFT_253833 Unclassified
348681957 hypothetical protein PHYSODRAFT_329682 Unclassified
348679629 Putative aldehyde reductase Unclassified
348677732 Hypothetical protein PHYSODRAFT_351217 Unclassified
348677176 Hypothetical protein PHYSODRAFT_544745 Unclassified
348676390 Hypothetical protein PHYSODRAFT_286458 Unclassified
348675944 Hypothetical protein PHYSODRAFT_286379 Unclassified
348675844 hypothetical protein PHYSODRAFT_561378 Unclassified
348675783 Hypothetical protein PHYSODRAFT_333832 Unclassified
348673781 Hypothetical protein PHYSODRAFT_354996 Unclassified
348671617 Putative endo-1,3-beta-glucanase Unclassified
348670901 Hypothetical protein PHYSODRAFT_520792 Unclassified
348670499 Hypothetical protein PHYSODRAFT_564545 Unclassified
348670494 Hypothetical protein PHYSODRAFT_318600 Unclassified
348669879 Hypothetical protein PHYSODRAFT_258871 Unclassified
348669733 Pleiotropic drug resistance protein ABC superfamily Unclassified
348667665 hypothetical protein PHYSODRAFT_340572 Unclassified
348664988 Hypothetical protein PHYSODRAFT_356224 Unclassified
262111960 Long-chain-fatty-acid-CoA ligase Unclassified
262111199 Conserved hypothetical protein Unclassified
262109829 Endoribonuclease L-PSP Unclassified
262108642 Zinc finger CDGSH domain-containing protein 1 Unclassified
262107467 Conserved hypothetical protein Unclassified
262107418 Elongation of very long chain fatty acids protein Unclassified
262106687 Cytochrome P450 Unclassified
262106295 ketol-acid reductoisomerase Unclassified
262105739 Cyclopropane-fatty-acyl-phospholipid synthase Unclassified
262104643 NmrA-like family protein Unclassified
262104423 conserved hypothetical protein Unclassified
262102846 Conserved hypothetical protein Unclassified
262102598 Conserved hypothetical protein Unclassified
262102403 Electron transfer flavoprotein subunit alpha Unclassified
262100267 mannitol dehydrogenase, putative Unclassified
262100233 Conserved hypothetical protein Unclassified
262099516 Estradiol 17-beta-dehydrogenase Unclassified
262098991 Endoplasmic reticulum-Golgi intermediate compartment protein Unclassified
262098869 conserved hypothetical protein Unclassified
262098739 Electron transfer flavoprotein subunit beta Unclassified
262097505 deoxyhypusine hydroxylase, putative Unclassified
262096965 Conserved hypothetical protein Unclassified
262096670 Conserved hypothetical protein Unclassified
262096466 Conserved hypothetical protein Unclassified
262096097 ATP-binding Cassette (ABC) Superfamily Unclassified
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Figure 1. GO annotation of candidate proteins associated with the adaptive response of P. capsici to flumorph.

Figure 1

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Numbers indicate the number of proteins categorized into each functional group.

Figure 2. Distribution of candidate proteins associated with the adaptive response of P. capsici to flumorph as categorized by KEGG pathway analysis.

Figure 2

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Numbers indicate the number of proteins in each category.

Discussion

An iTRAQ-LC-MS/MS approach was used to investigate the effect of the CAA fungicide flumorph on wild-type and resistant isolates of P. capsici. Altogether, 2406 individual proteins were identified, a number of 189 and 26 proteins were found to have altered levels of abundance in response to flumorph stress in PCAS1 and S2-838, respectively. One reason for the big difference in the number of differentially expressed proteins between the isolates PCAS1 and S2-838 can be contributed to the different genetic background. Compared to the wild-type isolate, the point mutations in cellulose synthase 3 caused the resistance to CAA fungicides in the mutant25,26. The 181 proteins related with genetic background of flumorph resistance were identified as candidates for the adaptive response of P. capsici to flumorph. The subsequent GO analysis categorized the proteins into 14 biological processes. However, KEGG pathway analysis indicated that 51 of the proteins have yet to be assigned metabolic pathways and therefore provide little insight into the effect of flumorph, although they could be utilized as candidate proteins for future study. The roles of the remaining 130 proteins were discussed below.

Carbohydrate metabolism was the pathway most affected by flumorph and was associated with the altered abundance of 46 proteins, of which 10 and 36 were up-regulated and down-regulated in response to flumorph, respectively. Two of the up-regulated proteins, Glucan-1,3-beta-glucosidase (Accession number: 262097763, and 262098611) and exo-1,3-beta-glucanase are involved in the break down glucan to release glucose27,28. Previous investigations into the mode of action of CAA fungicides have revealed that mandipropamid and pyrimorph can inhibit cell wall biosynthesis in P. infestans and P. capsici, respectively25,26. Given that the cell walls of oomycetes mainly consist of cellulose and 1,3-β-glucans29, the increased abundance of glucan-1,3-beta-glucosidase and exo-1,3-beta-glucanase suggested that P. capsici utilized carbohydrate from the host environment to compensate for the cell wall stress induced by flumorph. Similarly, the increased abundance of PHYSODRAFT_261542, PHYSODRAFT_302104, and PHYSODRAFT_565653, which are involved in glucan biosynthesis30, could also represent an adaptation to the cell wall stress induced by flumorph. The most down-regulated proteins were associated with glycolysis and the citric acid (TCA) cycle, which are involved in the utilization of glucose and other carbohydrates to generate ATP. However, interestingly, all the proteins involved in lipid metabolism, which can also result in the production of large amounts of energy, were up-regulated in response to flumorph. The altered level of these flumorph-responsive proteins suggested that flumorph might induce a redistribution of the metabolic processes associated with energy production. This hypothesized shift of energy generation from glycolysis and the citrate cycle to lipid metabolism could allow for the redistribution of glucose or carbohydrate in response to the inhibition of cell wall biosynthesis caused by flumorph.

Although the majority of the proteins with altered levels of abundance were associated with energy metabolism, a significant number of proteins (9 up-regulated, 20 down-regulated) were associated with amino acid metabolism. Several of the down-regulated proteins were found to play a role in the biosynthesis of amino acids and proteins including argininosuccinate lyase31 and the hypothetical protein PHYSODRAFT_48539930, which are involved in arginine biosynthesis; hypothetical protein PHYSODRAFT_47171330 and 5-methlytetrahydropteroyltriglutamate-homocysteine methyltransferease32, which play a role in methionine biosynthesis; and cysteine synthase, which participates in cysteine biosynthesis33; as well as the eukaryotic translation initiation factor 334, all of which might play important roles in protein synthesis. It is therefore possible that a reduced rate of global protein synthesis could be another adaptive response of P. capsici to flumorph stress as the pathogen attempts to maintain the fidelity of its protein biosynthesis. Similar results have also been observed in the response of P. capsici to another CAA fungicide, pyrimorph25.

It was also interesting that some proteins with altered levels of abundance were associated with response to stimulus and signal transduction. For example, alkaline phosphatase35 was up-regulated in response to flumorph. This important hydrolase enzyme catalyzes dephosphorylation during the post-translational modification of proteins36. Dephosphorylation and phosphorylation of S, T, Y and H residues are the best characterized modifications associated with the reversible, activation and inactivation of enzyme activity and the modulation of molecular interactions in signaling pathways37. It was also found that elicitin protein was up-regulated. Elicitin superfamily of proteins are structurally related to extracellular proteins that induce hypersensitive cell death and other biochemical changes associated with the defense response38,39,40. The up-regulation of alkaline phosphatase and elicitin in P. capsici therefore suggested that signal transduction was an important factor in responding to flumorph stress.

It was also found that a protein ABCA1 (from lipid exporter family) associated with transmembrane transport, an ATP binding cassette A (ABCA) superfamily protein was up-regulated in P. capsici in response to flumorph. Members of the ABCA family proteins have also been implicated in the adaptation to environmental changes in the free-living state of Phytophthora sojae41. The activation of this transporter could confer significant selective advantage to P. capsici isolates responding to flumorph stress in their environment.

Taken together, these results indicated that the adaptive response of P. capsici to flumorph was complex and regulated by multiple pathways, including utilising carbohydrate from the host environment to compensate for the cell wall stress induced by flumorph, a shift in energy generation from glycolysis and citrate cycle to lipid metabolism, decreased amino acids biosynthesis, and elevated levels of proteins associated with the pathogen’s response to stimulus and transmembrane transport. The proteomic data produced in the current study could provide important insight into the adaptive response of P. capsici to flumorph that would be useful for monitoring the emergence of resistance in field populations.

Material and Methods

Strains, medium, and growth conditions

The wild-type P. capsici isolate PCAS1 (P1314, mating type A1), which was originally collected from diseased green pepper (Capsicum annuum L.), was kindly provided by Professor Michael Coffey (University of California, Riverside, USA), while the sexual progeny S2-838 was generated in a previous study22. The EC50 values (the effective concentration for 50% inhibition of mycelial growth) for flumorph in the two isolates was approximately 1.5 μg/ml and 100 μg/ml (the maximal soluble concentration of flumorph), respectively (Supplementary Fig. S1). Potato dextrose agar (PDA) or potato dextrose broth (PDB) was used for routine maintenance of the cultures, which were dark incubated at 25 °C.

Sample preparation for proteomic analysis

Mycelium collected from 4-day cultures growing on PDA medium with a cellophane sheet were harvested and used to inoculate PDB medium in the presence or absence of flumorph (1.5 μg/ml or 100 μg/ml for the wild-type and resistant isolates, respectively). Each treatment has 10 biological replicates. After 24 hours dark-incubation at 25 °C with shaking, the mycelia were collected by filtration, washed profusely with sterile distilled water, dried, and ground thoroughly in liquid nitrogen. The resulting samples were stored at −80 °C until required. The experiment was repeated three times. The protein was extracted from approximately 100 μg of each sample, which were resuspended in 1 ml lysis buffer [8 M urea, 30 mM HEPES, 5 mM TCEP, and 2 mM EDTA] with the aid of a sonicator (Branson® Sonifier 250, BRANSON Ultrasonics Corporation, Danbury, U. S. A.). Any undisrupted cells were removed by centrifugation with the supernatant being transferred to fresh tubes. The samples were then incubated at 60 °C for one hour before the addition of 1% iodoethanol for another one hour in dark. The proteins were precipitated overnight in a freezer using 4 volumes of cold acetone, before being collected by centrifugation at 20000 rpm for 20 min. Finally, the proteins were dissolved in 50 mM triethylammonium bicarbonate (TEAB) containing 1% SDC.

Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) and Liquid Chromatography Coupled with Tandem Mass Spectrometry (LC−MS/MS) Analysis

Since technical variation of iTRAQ measurements was demonstrated to be on the order of 20%42, pooling samples were used to produce such biases43,44. In our study, ten biological replicates for each sample were pooled together to produce one sample, and 100 μg aliquots digested with 1 μg/μl trypsin overnight at 37 °C. After being lyophilized the samples belonging to individual treatments were labeled with different iTRAQ reagents (Applied Biosystems, Foster City, CA.) according to the protocol of the manufacturer. The untreated PCAS1 control was labeled with 114, while the PCAS1 treated with flumorph, S2-838 control and S2-838 treated with flumorph were labeled with 115, 116 and 117, respectively. The labeled peptides were then combined and dried in a vacuum concentrator. The first dimension of the 2D-LC consisted of extensive fractionation of the peptide mixtures by strong cation exchange (SCX) chromatography to improve proteome coverage. Briefly, the dried samples were reconstituted in buffer A [25% (v/v) acetonitrile (ACN), 10 mM potassium phosphate; pH adjusted to 3.0] and loaded onto a Lumn A column (4.6-mm i.d. × 100-mm length, 5 μm, 100 Å; Phenomenex, USA). The column was equilibrated for 10 min in buffer A before the peptides were eluted at a flow rate of 1 mL/min using buffer B [25% (v/v) ACN, 2 M potassium chloride, 10 mM potassium phosphate; pH adjusted to 3] at a succession of increasing gradients 0–30% for 15 min, followed by 30–100% for 15 min, and finally 100% buffer B for 10 min. A total of fifteen peptide fractions were collected, which were then dried using a SpeedVac centrifugal vacuum concentrator and purified on a strata-X C18 column (Phenomenex, USA) prior to mass spectrometry (MS) analysis.

The LC-MS/MS experiments were performed using an integrated system consisting of a Q Exactive™ Mass Spectrometer (Thermo Fisher Scientific, USA) coupled with a nanoflow HPLC system (Easy nLC, Proxeon Biosystems, now Thermo Fisher Scientific, USA). Each fraction was reconstituted in 0.1% formic acid before being injected into the LC-MS/MS system. The samples eluted from the trap column were separated on a PepMap C18 column (100 mm × 75 mm, 300-Å pore size, 5 μm particle size, Thermo scientific, USA) at a rate of 400 nL/min, using 0.1% formic acid as solvent A and 0.1% formic acid in acetonitrile as solvent B, in increasing gradients: 0.1–5% B (0–10 min), 5–30% B (10–40 min), 30–60% B (40–45 min), 60–80% B (45–48 min), 80% B (48–55 min), 80–0.1% B (55–65 min). The eluting peptides were sprayed into the mass spectrometer at an ion spray voltage of 1800 eV, and their MS/MS spectra acquired using automated data-directed switching between the MS and MS/MS modes. The five most abundant signals from each survey scan (350–2000 m/z range) were selected by charge state, and the collision energy applied accordingly for the sequential MS/MS fragmentation scanning as described previously45. The entire experiment was conducted three times.

Data Processing and Analysis

The raw MS/MS data were merged and transformed using the Proteome Discoverer software package (version 1.3; Thermo Fisher Scientific, USA)46 before Mascot version 2.3.01 (Matrix Sciences, Ltd., London, UK) was used to identify and quantify the individual proteins according to sequences contained in the NCBI Oomycetes database using the following settings: trypsin specific digestion with one missed cleavage allowed, peptide tolerance of 15 ppm, MS/MS tolerance of 20 mmu, iTRAQ 4-plex for peptide N-t and Lys as fixed modifications, and in variable mode, iTRAQ 4-plex on Tyr, oxidized Met and methylthio on Cys. The false positive rate, which was checked using a concatenated target-decoy database search strategy, was set to be less than 1%. Only proteins with at least one unique peptide and having protein scores of more than 20 were initially recorded. Only proteins with two or more peptides were used for the quantitative analysis. The LIBRA tool from the TPP software47 was used for protein quantification using the default parameters. The relative abundance of proteins in the different treatments were calculated from three replicates using the log2 of the iTRAQ ratios, which were normalized before the standard deviations from the corresponding normal distributions of ratios were used to determine the cutoff point of the experiment48. Proteins whose average ratios fell outside a standard deviation of ±1 from the global mean were considered to have differential abundance. Gene Ontology (GO) annotation was conducted using information retrieved from the UniProt and BGI WEGO (http://wego.genomics.org.cn) databases49, while the pathway enrichment analysis was conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway database50.

Additional Information

How to cite this article: Pang, Z. et al. Insights into the adaptive response of the plant-pathogenic oomycete Phytophthora capsici to the fungicide flumorph. Sci. Rep. 6, 24103; doi: 10.1038/srep24103 (2016).

Supplementary Material

Supplementary Information
srep24103-s1.pdf (98.9KB, pdf)
Supplementary Table S1
srep24103-s2.xls (511.5KB, xls)
Supplementary Table S2
srep24103-s3.xls (18.5MB, xls)
Supplementary Table S3
srep24103-s4.xls (75.5KB, xls)
Supplementary Table S4
srep24103-s5.xls (25KB, xls)

Acknowledgments

This work was partially funded by the National Science Foundation of China (31272061), and the special Fund for Agro-scientific Research in the Public Interest (201303023, 201203022).

Footnotes

Author Contributions Z.P. performed the whole experiment; L.C. and X.L. conceived the experiment; Z.P. and X.L. wrote the paper.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Information
srep24103-s1.pdf (98.9KB, pdf)
Supplementary Table S1
srep24103-s2.xls (511.5KB, xls)
Supplementary Table S2
srep24103-s3.xls (18.5MB, xls)
Supplementary Table S3
srep24103-s4.xls (75.5KB, xls)
Supplementary Table S4
srep24103-s5.xls (25KB, xls)

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