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Fungicides are an indispensable tool in plant disease control. Various modes of action (MOAs) have been identified in different fungicides to suppress plant pathogens. The combined use of fungicides with distinct MOAs has been recommended to prevent the development of pathogen resistance. In studying MOAs, metabolomics has been proven to be a robust and high-throughput method. Because metabolites are unique and distinct depending on the biological activities of an organism, MOAs can be identified and classified by establishing metabolic fingerprinting and metabolic profiles. Similarly, if fungicide resistance is developed in a pathogen, the metabolome will change, which can be identified. In this review, we have discussed the principles and advanced applications of metabolomics in the study of MOAs and resistance mechanisms of fungicides, and the potential of metabolic data in understanding the interaction between fungicides and pathogens. Challenges are also discussed in the application of metabolomics, improvement of the study on the mechanism of fungicides in their functions against pathogens and advancing the development of novel fungicides.

Abstract Host-derived reactive oxygen species (ROS) are an important defense means to protect against pathogens. Although mitochondria are the main intracellular targets of ROS, how pathogens regulate mitochondrial physiology in response to oxidative stress remains elusive. Prohibitin 2 (PHB2) is an inner mitochondrial membrane (IMM) protein, recognized as a mitophagy receptor in animals and fungi. Here, we find that an ANK and FYVE domain-containing protein PsAF5, is an adapter of PsPHB2, interacting with PsATG8 under ROS stress. Unlike animal PHB2 that can recruit ATG8 directly to mitochondria, PsPHB2 in Phytophthora sojae cannot recruit PsATG8 to stressed mitochondria without PsAF5. PsAF5 deletion impairs mitophagy under ROS stress and increases the pathogen’s sensitivity to H2O2, resulting in the attenuation of P. sojae virulence. This discovery of a PsPHB2-PsATG8 adapter (PsAF5) in plant-pathogenic oomycetes reveals that mitophagy induction by IMM proteins is conserved in eukaryotes, but with differences in the details of ATG8 recruitment.

Small RNAs (sRNAs) are important non-coding RNA regulators that play key roles in the development and pathogenesis of plant pathogens, as well as in other biological processes. However, whether these abundant and varying sRNAs are involved in Phytophthora development or infection remains enigmatic. In this study, sRNA sequencing of 4 asexual stages of Phytophthora capsici (P. capsici), namely, as mycelia (HY), sporangia (SP), zoospores (ZO), cysts (CY), and pepper infected with P. capsici (IN), were performed, followed by sRNA analysis, microRNA (miRNA) identification, and miRNA target prediction. sRNAs were mainly distributed at 25–26 nt in HY, SP, and ZO but distributed at 18–34 nt in CY and IN. 92, 42, 176, 39, and 148 known miRNAs and 15, 19, 54, 13, and 1 novel miRNA were identified in HY, SP, ZO, CY, and IN, respectively. It was found that the expression profiles of known miRNAs vary greatly at different stages and could be divided into 4 categories. Novel miRNAs mostly belong to part I. Gene ontology (GO) analysis of known miRNA-targeting genes showed that they are involved in the catalytic activity pathway, binding function, and other biological processes. Kyoto Encyclopedia of Gene and Genome (KEGG) analysis of novel miRNA-targeting genes showed that they are involved in the lysine degradation pathway. The expression of candidate miRNAs was validated using quantitative reverse transcription–polymerase chain reaction (qRT–PCR), and miRNAs were downregulated in PcDCL1 or PcAGO1 mutants. To further explore the function of the detected miRNAs, the precursor of a novel miRNA, miR91, was knockout by CRISPR-Cas9, the mutants displayed decreased mycelial growth, sporangia production, and zoospore production. It was found that 503142 (Inositol polyphosphate 5-phosphatase and related proteins) can be predicted as a target of miR91, and the interaction between miR91 and 503142 was verified using the tobacco transient expression system. Overall, our results indicate that the diverse and differentially expressed sRNAs are involved in the development and pathogenesis of P. capsici.

Proper transcription orchestrated by RNA polymerase II (RNPII) is crucial for cellular development, which is rely on the phosphorylation state of RNPII's carboxyl-terminal domain (CTD). Sporangia, developed from mycelia, are essential for the destructive oomycetes Phytophthora, remarkable transcriptional changes are observed during the morphological transition. However, how these changes are rapidly triggered and their relationship with the versatile RNPII-CTD phosphorylation remain enigmatic. Herein, we found that Phytophthora capsici undergone an elevation of Ser5-phosphorylation in its uncanonical heptapeptide repeats of RNPII-CTD during sporangia development, which subsequently changed the chromosomal occupation of RNPII and primarily activated transcription of certain genes. A cyclin-dependent kinase, PcCDK7, was highly induced and phosphorylated RNPII-CTD during this morphological transition. Mechanistically, a novel DCL1-dependent microRNA, pcamiR1, was found to be a feedback modulator for the precise phosphorylation of RNPII-CTD by complexing with PcAGO1 and regulating the accumulation of PcCDK7. Moreover, this study revealed that the pcamiR1-CDK7-RNPII regulatory module is evolutionarily conserved and the impairment of the balance between pcamiR1 and PcCDK7 could efficiently reduce growth and virulence of P. capsici. Collectively, this study uncovers a novel and evolutionary conserved mechanism of transcription regulation which could facilitate correct development and identifies pcamiR1 as a promising target for disease control.

Abstract A large number of La-related proteins (LARPs), most of which share a La-motif and one or more adjacent RNA-recognition (RRM) domains, are known to play a function in diverse processes. Among the LAPRs, LARP4 and LARP5 have been mainly reported to act as positive translation factors. In Phytophthora sojae, only one protein with typical LARP protein features was identified, and it was named LARP4_5 due to the presence of an RRM_LARP4_5-like domain. The PsLARP4_5 gene was significantly upregulated in zoospores and during the infection stage. By comparing the biological characteristics of a wild-type strain with three PsLARP4_5 knockout transformants, it was found that PsLARP4_5 was involved in mycelial growth, sporangium and zoospore production, and pathogenicity of P. sojae. Further analysis of the transcriptome indicated that many differentially expressed genes could participate in several essential biological processes in the PsLARP4_5 transformant, including translation, as structural constituents of ribosomes or cytosolic large ribosomal subunits and others. Notably, 76 genes with a role in the ribosome pathway were downregulated in the PsLARP4_5 transformant, suggesting that PsLARP4_5 might affect translation. Overall, these findings indicate that PsLARP4_5 plays an essential role in the development and pathogenicity of P. sojae.

ABSTRACT Multi-fungicide resistance (MFR) is a serious environmental problem, which results in the excessive use of fungicides. Fitness penalty, as a common phenomenon in MFR, can partially counteract the issue of resistance due to the weakened vigor of MFR pathogens. Their underlying mechanism and relationship remain unexplained. By Oxford Nanopore Technologies sequencing and dot blot, we found that N6-methyloxyadenine (6mA) modification, the dominate epigenetic marker in Phytophthora capsici, was significantly altered after MFR emerged. Among the differently methylated genes, PcGSTZ1 could efficiently detoxify SYP-14288, a novel uncoupler, through complexing the fungicide with glutathione and induce MFR. Interestingly, PcGSTZ1 overexpression was induced by elevated 6mA levels and chromatin accessibility to its genomic loci. Moreover, the overexpression led to reactive oxygen species burst and ferroptosis in SYP-14288-resistant mutants, which enhanced the resistance and induced fitness penalty in P. capsici through triggering low energy shock adaptive response. Furthermore, this study revealed that the 6mA-PcGSTZ1-ferroptosis axis could mediate intergenerational resistance memory transmission and enabled adaptive advantage to P. capsici. In conclusion, the findings provide new insights into the biological role of 6mA as well as the mechanisms underlying the trade-off between MFR and fitness. These could also benefit disease control through the blockade of the epigenetic axis to resensitize resistant isolates.IMPORTANCEN6-methyloxyadenine (6mA) modification on DNA is correlated with tolerance under different stress in prokaryotes. However, the role of 6mA in eukaryotes remains poorly understood. Our current study reveals that DNA adenine methyltransferase 1 (DAMT1)-mediated 6mA modification at the upstream region of GST zeta 1 (GSTZ1) is elevated in the resistant strain. This elevation promotes the detoxification uncoupler and induces multifungicide resistance (MFR). Moreover, the overexpression led to reactive oxygen species burst and ferroptosis in SYP-14288-resistant mutants, which enhanced the resistance and induced fitness penalty in Phytophthora capsici through triggering low energy shock adaptive response. Furthermore, this study revealed that the 6mA-PcGSTZ1-ferroptosis axis could mediate intergenerational resistance memory transmission and enabled adaptive advantage to P. capsici. Overall, our findings uncover an innovative mechanism underlying 6mA modification in regulating PcGSTZ1 transcription and the ferroptosis pathway in P. capsici.

ABSTRACT Fungicides are an effective way to control gray mold of grapes, but the pathogen Botrytis cinerea can develop resistance, overcoming the effectiveness of a fungicide that is repeatedly applied. More importantly, the emergence of multidrug resistance (MDR) in the field, where multiple fungicides with different modes of action simultaneously lose their efficacies, is a significant concern. MDR is associated with ATP-binding cassette (ABC) transporters of the pathogen, and certain plant secondary metabolites (PSMs) stimulate the upregulation of ABC transporters, we hypothesized that the pathogen’s preadaptation to PSMs might contribute to MDR development. To test this in B. cinerea, ten PSMs, namely, resveratrol, reserpine, chalcone, flavanone, eugenol, farnesol, anethene, camptothecin, salicylic acid, and psoralen, were selected based on their association with ABC transporters involved in fungicide resistance. B. cinerea strain B05.10 was continuously transferred for 15 generations on potato dextrose agar amended with a PSM (PDAP), and sensitivities to PSMs and fungicides were examined on the 5th, 10th, and 15th generations. RNA was extracted from B. cinerea from the selected generations. After 15 generations of culture transfers, an up-regulation was observed in the expression of ABC transporter-encoding genes BcatrB, BcatrD, and BcatrK using quantitative polymerase chain reaction (qPCR). This upregulation was found to contribute to MDR of B. cinerea against two or more fungicides, among azoxystrobin, boscalid, fludioxonil, difenoconazole, prochloraz, and pyrimethanil. This finding was confirmed through genetic transformation. The decreased sensitivity of B. cinerea to fungicides was confirmed as a subsequent MDR phenotype after exposure to camptothecin, flavanone, and resveratrol. Besides, transcriptome analysis also revealed the upregulation of transcription factors related to ABC expression following resveratrol exposure. This suggests that PSMs contributed to inducing preadaptation of B. cinerea, leading to subsequent MDR.IMPORTANCEThe emergence of MDR in plant pathogens is a threat to plant disease management and leads to the use of excessive fungicides. Botrytis cinerea is of particular concern because its MDR has widely emerged in the field. Understanding its genesis is the first step for controlling MDR. In this study, the contribution of PSMs to MDR has been examined. Effective management of this pathogen in agroecosystems relies on a better understanding of how it copes with phytochemicals or fungicides.

To achieve a stable size distribution over multiple generations, proliferating cells require a means of counteracting stochastic noise in the rate of growth, the time spent in various phases of the cell cycle, and the imprecision in the placement of the plane of cell division. In the most widely accepted model, cell size is thought to be regulated at the G1/S transition, such that cells smaller than a critical size pause at the end of G1 phase until they have accumulated mass to a predetermined size threshold, at which point the cells proceed through the rest of the cell cycle. However, a model, based solely on a specific size checkpoint at G1/S, cannot readily explain why cells with deficient G1/S control mechanisms are still able to maintain a very stable cell size distribution. Furthermore, such a model would not easily account for stochastic variation in cell size during the subsequent phases of the cell cycle, which cannot be anticipated at G1/S. To address such questions, we applied computationally enhanced quantitative phase microscopy (ceQPM) to populations of cultured human cell lines, which enables highly accurate measurement of cell dry mass of individual cells throughout the cell cycle. From these measurements, we have evaluated the factors that contribute to maintaining cell mass homeostasis at any point in the cell cycle. Our findings reveal that cell mass homeostasis is accurately maintained, despite disruptions to the normal G1/S machinery or perturbations in the rate of cell growth. Control of cell mass is generally not confined to regulation of the G1 length. Instead mass homeostasis is imposed throughout the cell cycle. In the cell lines examined, we find that the coefficient of variation (CV) in dry mass of cells in the population begins to decline well before the G1/S transition and continues to decline throughout S and G2 phases. Among the different cell types tested, the detailed response of cell growth rate to cell mass differs. However, in general, when it falls below that for exponential growth, the natural increase in the CV of cell mass is effectively constrained. We find that both mass-dependent cell cycle regulation and mass-dependent growth rate modulation contribute to reducing cell mass variation within the population. Through the interplay and coordination of these 2 processes, accurate cell mass homeostasis emerges. Such findings reveal previously unappreciated and very general principles of cell size control in proliferating cells. These same regulatory processes might also be operative in terminally differentiated cells. Further quantitative dynamical studies should lead to a better understanding of the underlying molecular mechanisms of cell size control.

The necrotrophic pathogen Botrytis cinerea infects a broad range of plant hosts and causes substantial economic losses to many crops. Although resistance to procymidone has been observed in the field, it remains uncertain why procymidone is usually involved in multidrug resistance (MDR) together with other fungicides. Nine mutants derived from the B. cinerea strain B05.10 through procymidone domestication exhibited high resistance factors (RFs) against both procymidone and fludioxonil. However, the fitness of the mutants was reduced compared to their parental strain, showing non-sporulation and moderate virulence. Furthermore, the RFs of these mutants to other fungicides, such as azoxystrobin, fluazinam, difenoconazole, and pyrimethanil, ranged from 10 to 151, indicating the occurrence of MDR. Transcriptive expression analysis using the quantitative polymerase chain reaction (qPCR) revealed that the mutants overexpressed ABC transporter genes, ranging from 2 to 93.7-fold. These mutants carried single-point mutations W647X, R96X, and Q751X within BcBos1 by DNA sequencing. These alterations in BcBos1 conferred resistance to procymidone and other fungicides in the mutants. Molecular docking analysis suggested distinct interactions between procymidone and Bos1 in the B. cinerea standard strain B05.10 or the resistant mutants, suggesting a higher affinity of the former towards binding with the fungicide. This study provides a comprehensive understanding of the biological characteristics of the resistant mutants and conducts an initial investigation into its fungicide resistance traits, providing a reference for understanding the causes of multidrug resistance of B. cinerea in the field.

Abstract Greenhouse-grown cucumbers were monitored over two successive growing seasons to investigate the effects of successive application of ZJ0712, a new quinone outside inhibitor (QoI) fungicide, on the development of resistance in cucumber powdery mildew (Podosphaera xanthii). Resistant P. xanthii isolates were detected after nine successive applications of ZJ0712, although the control efficacy of this fungicide against cucumber powdery mildew at that time was still higher than 80%. Seven ZJ0712-resistant P. xanthii isolates with resistance factor values greater than 180 were obtained, which exhibited a stable resistance to ZJ0712. These resistant P. xanthii isolates had similar pathogenicity to the wild-type isolate on cucumber plants. The ZJ0712 showed significant cross-resistance with azoxystrobin, enostrobilurin, or chlorothalonil, but not with the azole fungicide triadimefon. Furthermore, the most commonly reported G143A mutation in Cyt b associated with QoI resistance was found in five of the seven resistant isolates. These findings suggest that there is a high risk of resistance development associated with using ZJ0712 for controlling cucumber powdery mildew under greenhouse conditions, and the underlying resistance mechanisms in different P. xanthii isolates are not consistent and need to be further unraveled.

ABSTRACT Phytophthora species are devastating filamentous plant pathogens that belong to oomycetes, a group of microorganisms similar to fungi in morphology but phylogenetically distinct. They are sterol auxotrophic, but nevertheless exploit exogenous sterols for growth and development. However, as for now the mechanisms underlying sterol utilization in Phytophthora are unknown. In this study, we identified four genes in Phytophthora capsici that encode proteins containing a sterol-sensing domain (SSD), a protein domain of around 180 amino acids comprising five transmembrane segments and known to feature in sterol signaling in animals. Using a modified CRISPR/Cas9 system, we successfully knocked out the four genes named PcSCP1 to PcSCP4 (for P. capsici SSD-containing protein 1 to 4), either individually or sequentially, thereby creating single, double, triple, and quadruple knockout transformants. Results showed that knocking out just one of the four PcSCPs was not sufficient to block sterol signaling. However, the quadruple “all-four” PcSCPs knockout transformants no longer responded to sterol treatment in asexual reproduction, in contrast to wild-type P. capsici that produced zoospores under sterol treatment. Apparently, the four PcSCPs play a key role in sterol signaling in P. capsici with functional redundancy. Transcriptome analysis indicated that the expression of a subset of genes is regulated by exogenous sterols via PcSCPs. Further investigations showed that sterols could stimulate zoospore differentiation via PcSCPs by controlling actin-mediated membrane trafficking. Moreover, the pathogenicity of the “all-four” PcSCPs knockout transformants was significantly decreased and many pathogenicity related genes were downregulated, implying that PcSCPs also contribute to plant-pathogen interaction. IMPORTANCE Phytophthora is an important genus of oomycetes that comprises many destructive plant pathogens. Due to the incompleteness of the sterol synthesis pathway, Phytophthora spp. do not possess the ability to produce sterols. Therefore, these sterol auxotrophic oomycetes need to recruit sterols from the environment such as host plants to support growth and development, which seems crucial during pathogen-plant interactions. However, the mechanisms underlying sterol utilization by Phytophthora spp. remain largely unknown. Here, we show that a family of sterol-sensing domain-containing proteins (SCPs) consisting of four members in P. capsici plays a key role in sterol signaling with functional redundancy. Moreover, these SCPs play a role in different biological processes, including asexual reproduction and pathogenicity. Our study overall revealed the multiple functions of PcSCPs and addressed the question of how exogenous sterols regulate the development of heterothallic Phytophthora spp. via SSD-containing proteins.

Guvermectin is a recently discovered microbial N9-glucoside cytokinin compound extracted from Streptomyces sanjiangensis NEAU6. Although some research has reported that N9-glucoside cytokinin compounds do not have the activity of cytokinin, it has been noted that guvermectin can promote growth and antifungal activity in Arabidopsis. Maize is an important food crop in the world and exploring the effect of guvermectin on this crop could help its cultivation in regions with adverse environmental conditions such as a high temperature. Here, we investigated the effects of guvermectin seed soaking treatment on the growth of maize at the seedlings stage and its yield attributes with different temperature stresses. The maize (cv. Zhengdan 958) with guvermectin seed soaking treatment were in two systems: paper roll culture and field conditions. Guvermectin seed soaking treated plants had increased plant height, root length, and mesocotyl length at the seedlings stage, and spike weight at maturity in the field. But only root length was increased at the paper roll culture by guvermectin seed soaking treatment. Guvermectin seed soaking treatment reduced the adverse effects on maize seedling when grow at a high temperature. Further experiments showed that, in high temperature conditions, guvermectin treatment promoted the accumulation of heat shock protein (HSP) 17.0, HSP 17.4 and HSP 17.9 in maize roots. Comparative transcriptomic profiling showed there were 33 common differentially expressed genes (DEGs) in guvermectin treated plants under high temperature and room temperature conditions. The DEGs suggested that guvermectin treatment led to the differential modulation of several transcripts mainly related with plant defense, stress response, and terpenoid biosynthesis. Taken together, these results suggested that the guvermectin treatment promoted the growth and tolerance of high temperature stresses, possibly by activation of related pathways. These results show that guvermectin is a novel plant growth regulator and could be developed as an application to maize seeds to promote growth in high temperature environments.

Cell dry mass is principally determined by the sum of biosynthesis and degradation. Measurable change in dry mass occurs on a time scale of hours. By contrast, cell volume can change in minutes by altering the osmotic conditions. How changes in dry mass and volume are coupled is a fundamental question in cell size control. If cell volume were proportional to cell dry mass during growth, the cell would always maintain the same cellular mass density, defined as cell dry mass dividing by cell volume. The accuracy and stability against perturbation of this proportionality has never been stringently tested. Normalized Raman Imaging (NoRI), can measure both protein and lipid dry mass density directly. Using this new technique, we have been able to investigate the stability of mass density in response to pharmaceutical and physiological perturbations in three cultured mammalian cell lines. We find a remarkably narrow mass density distribution within cells, that is, significantly tighter than the variability of mass or volume distribution. The measured mass density is independent of the cell cycle. We find that mass density can be modulated directly by extracellular osmolytes or by disruptions of the cytoskeleton. Yet, mass density is surprisingly resistant to pharmacological perturbations of protein synthesis or protein degradation, suggesting there must be some form of feedback control to maintain the homeostasis of mass density when mass is altered. By contrast, physiological perturbations such as starvation or senescence induce significant shifts in mass density. We have begun to shed light on how and why cell mass density remains fixed against some perturbations and yet is sensitive during transitions in physiological state.

Proteins containing both FYVE and serine/threonine kinase catalytic (STKc) domains are exclusive to protists. However, the biological function of these proteins in oomycetes has rarely been reported. In the Phytophthora sojae genome database, we identified five proteins containing FYVE and STKc domains, which we named PsZFPK1, PsZFPK2, PsZFPK3, PsZFPK4, and PsZFPK5. In this study, we characterized the biological function of PsZFPK1 using a CRISPR/Cas9-mediated gene replacement system. Compared with the wild-type strain, P6497, the PsZFPK1-knockout mutants exhibited significantly reduced growth on a nutrient-rich V8 medium, while a more pronounced defect was observed on a nutrient-poor Plich medium. The PsZFPK1-knockout mutants also showed a significant increase in sporangium production. Furthermore, PsZFPK1 was found to be essential for oospore production and complete virulence but dispensable for the stress response in P. sojae. The N-terminal region, FYVE and STKc domains, and T602 phosphorylation site were found to be vital for the function of PsZFPK1. Conversely, these domains were not required for the localization of PsZFPK1 protein in the cytoplasm. Our results demonstrate that PsZFPK1 plays a critical role in vegetative growth, sporangium formation, oospore production, and virulence in P. sojae.

In response to pathogen infection, some plants increase production of secondary metabolites, which not only enhance plant defense but also induce fungicide resistance, especially multidrug resistance (MDR) in the pathogen through preadaptation. To investigate the cause of MDR in Botrytis cinerea, grapes ‘Victoria’ (susceptible to B. cinerea) and ‘Shine Muscat’ (resistant to B. cinerea) were inoculated into seedling leaves with B. cinerea, followed by extraction of metabolites from the leaves on days 3, 6, and 9 after inoculation. The extract was analyzed using gas chromatography/quadrupole time-of-flight mass (GC/QTOF) combined with solid-phase microextraction (SPME) for volatile and nonvolatile metabolomic components. Nonvolatile metabolites γ-aminobutyric acid (GABA), resveratrol, piceid, and some carbohydrates or amino acids, coupled with volatile metabolites β-ocimene, α-farnesene, caryophyllene, germacrene D, β-copaene, and alkanes, accumulated at a higher level in grape leaves infected with B. cinerea compared to in noninoculated leaves. Among the established metabolic pathways, seven had greater impacts, including aminoacyl-tRNA biosynthesis, galactose metabolism, valine, leucine, and isoleucine biosynthesis. Furthermore, isoquinoline alkaloid biosynthesis; phenylpropanoid biosynthesis; monobactam biosynthesis; tropane, piperidine, and pyridine alkaloid biosynthesis; phenylalanine metabolism; and glucosinolate biosynthesis were related to antifungal activities. Based on liquid chromatography/quadrupole time-of-flight mass (LC/QTOF) detection and bioassay, B. cinerea infection induced production of plant secondary metabolites (PSMs) including eugenol, flavanone, reserpine, resveratrol, and salicylic acid, which all have inhibitory activity against B. cinerea. These compounds also promoted overexpression of ATP-binding cassette (ABC) transporter genes, which are involved in induction of MDR in B. cinerea.

Asparagine (Asn, N)-linked glycosylation is a conserved process and an essential post-translational modification that occurs on the NXT/S motif of the nascent polypeptides in endoplasmic reticulum (ER). The mechanism of N-glycosylation and biological functions of key catalytic enzymes involved in this process are rarely documented for oomycetes. In this study, an N-glycosylation inhibitor tunicamycin (TM) hampered the mycelial growth, sporangial release, and zoospore production of Phytophthora capsici, indicating that N-glycosylation was crucial for oomycete growth development. Among the key catalytic enzymes involved in N-glycosylation, the PcSTT3B gene was characterized by its functions in P. capsici. As a core subunit of the oligosaccharyltransferase (OST) complex, the staurosporine and temperature sensive 3B (STT3B) subunit were critical for the catalytic activity of OST. The PcSTT3B gene has catalytic activity and is highly conservative in P. capsici. By using a CRISPR/Cas9-mediated gene replacement system to delete the PcSTT3B gene, the transformants impaired mycelial growth, sporangial release, zoospore production, and virulence. The PcSTT3B-deleted transformants were more sensitive to an ER stress inducer TM and display low glycoprotein content in the mycelia, suggesting that PcSTT3B was associated with ER stress responses and N-glycosylation. Therefore, PcSTT3B was involved in the development, pathogenicity, and N-glycosylation of P. capsici.

Abstract Flumorph is a carboxylic acid amide (CAA) fungicide with high activity against oomycetes. However, evolution to CAAs from low resistance to high resistance has never been reported. This study investigated the basis of resistance evolution of flumorph in Phytophthora sojae. Total of 120 P. sojae isolates were collected and their sensitivity to flumorph was evaluated. Although no spontaneous resistance was found among the field isolates, adaptation on flumorph‐amended media resulted in the selection of five stable mutant types exhibiting varying degrees of resistance to CAAs. Type I, which exhibited the lowest resistance level, was obtained when the wild‐type isolate was exposed to a low concentration of flumorph, but no resistant mutants were obtained by direct exposure to higher concentrations. However, the more resistant types (Type II, III, IV and V) were obtained when Type I were exposed to higher concentrations of flumorph. Similar results were obtained when the entire screening process was repeated, which implied that evolution of resistance to flumorph in P. sojae could be a two‐step process, where high resistance phenotypes could develop gradually from low resistance ones. Further investigation into molecular mechanism strongly confirmed that evolution of isolates highly resistant to flumorph occurs in a stepwise process with Type I as intermediary, through accumulation of mutations in their target protein of CAAs (CesA3). Together, our findings indicate that application of low rates of flumorph in field could result in selection of low resistance Type I isolates, but that raising dosage to maintain comparable levels of control could elicit rapid evolution of more resistant Type II, III, IV and V isolates with stepwise accumulation of mutations in CesA3, which would render flumorph ineffective as a control method. Precautionary resistance management strategy should be implemented. The phenomenon described in the study could have broader biological significance.

Although sterols play an important role in most eukaryotes, some oomycetes, including Phytophthora spp., have lost the sterol synthesis pathway. Nevertheless, the ERG3 gene encoding C-5 sterol desaturase in the sterol synthesis pathway is still present in the genomes of Phytophthora spp. Phytophthora capsici, a destructive pathogen with a broad range of plant hosts, poses a significant threat to the production of agriculture. This study focused on the ERG3 gene in P. capsici (PcERG3) and explored its function in this pathogen. It showed that the PcERG3 gene could be expressed in all tested developmental stages of P. capsici, with sporangium and mycelium displaying higher expression levels. A potential substrate of Erg3 (stellasterol) was used to treat the P. capsici wild-type strain and a PcERG3Δ transformant, and their sterol profiles were determined by GC-MS. The wild-type strain could convert stellasterol into the down-stream product while the transformant could not, indicating that PcErg3 retains the C-5 sterol desaturase activity. By comparing the biological characteristics of different strains, it was found that PcERG3 is not important for the development of P. capsici. The pathogenicity of the PcERG3Δ transformants and the wild-type strain was comparable, suggesting that PcERG3 is not necessary for the interaction between P. capsici and its hosts. Further investigations revealed that the PcERG3Δ transformants and the wild-type strain displayed a similar level of tolerance to external adversities such as unsuitable temperatures, high osmotic pressures, and intemperate pH, signifying that PcERG3 is not essential for P. capsici to cope with these environmental stresses.

The de novo biosynthesis of sterols is critical for the majority of eukaryotes; however, some organisms lack this pathway, including most oomycetes. Phytophthora spp. are sterol auxotrophic but, remarkably, have retained a few genes encoding enzymes in the sterol biosynthesis pathway. Here, we show that PcDHCR7, a gene in Phytophthora capsici predicted to encode Δ7-sterol reductase, displays multiple functions. When expressed in Saccharomyces cerevisiae, PcDHCR7 showed the Δ7-sterol reductase activity. Knocking out PcDHCR7 in P. capsici resulted in loss of the capacity to transform ergosterol into brassicasterol, which means PcDHCR7 has the Δ7-sterol reductase activity in P. capsici itself. This enables P. capsici to transform sterols recruited from the environment for better use. The biological characteristics of ΔPcDHCR7 transformants were compared with those of the wild-type strain and a PcDHCR7 complemented transformant, and the results showed that PcDHCR7 plays a key role in mycelium development and pathogenicity of zoospores. Further analysis of the transcriptome indicated that the expression of many genes changed in the ΔPcDHCR7 transformant, which involve in different biological processes. It is possible that P. capsici compensates for the defects caused by the loss of PcDHCR7 by remodelling its transcriptome.

SYP-14288 is a fungicide as an uncoupler of oxidative phosphorylation, which is effective in controlling fungal pathogens like Rhizoctonia solani. To determine whether R. solani can develop SYP-14288 resistance and possibly multi-drug resistance (MDR), an SYP-14288-resistant mutant of R. solani X19-7 was generated from wild-type strain X19, and the mechanism of resistance was studied through metabolic and genetic assays. From metabolites of R. solani treated with SYP-14288, three compounds including M1, M2, and M3 were identified according to UPLC-MS/MS analysis, and M1 accumulated faster than M2 and M3 in X19-7. When X19-7 was treated by glutathione-S-transferase (GST) inhibitor diethyl maleate (DEM) and SYP-14288 together, or by DEM plus one of tested fungicides that have different modes of action, a synergistic activity of resistance occurred, implying that GSTs promoted metabolic resistance against SYP-14288 and therefore led to MDR. By comparing RNA sequences between X19-7 and X19, six cytochrome P450s (P450s) and two GST genes were selected as a target, which showed a higher expression in X19-7 than X19 both before and after the exposure to SYP-14288. Furthermore, heterologous expression of P450 and GST genes in yeast was conducted to confirm genes involved in metabolic resistance. In results, the P450 gene AG1IA_05136 and GST gene AG1IA_07383 were related to fungal resistance to multiple fungicides including SYP-14288, fluazinam, chlorothalonil, and difenoconazole. It was the first report that metabolic resistance of R. solani to uncouplers was associated with P450 and GST genes.

Extracellular vesicles (EVs) represent a prominent mechanism of transport and interaction between cells, especially microbes. Increasing evidence indicates that EVs play a key role in the physiological and pathological processes of pathogens and other symbionts. Recent research has focused on the specific functions of these vesicles during pathogen-host interactions, including trans-kingdom delivery of small RNAs, proteins and metabolites. Much current research on the function of EVs is focused on immunity and the interactions of microbes with human cells, while the roles of EVs during plant-microbe interactions have recently emerged in importance. In this review, we summarize recent research on the biogenesis of these vesicles and their functions in biology and pathology. Many key questions remain unclear, including the full structural and functional diversity of EVs, the roles of EVs in communication among microbes within microbiomes, how specific cargoes are targeted to EVs, whether EVs are targeted to specific destinations, and the full scope of EVs’ transport of virulence effectors and of RNA and DNA molecules.

Abstract Fungal pathogen Botrytis cinerea, the casual agent of gray mold of vegetables and fruits, has a high risk of developing resistance to fungicide. Tebuconazole, one kind of demethylation inhibitor (DMI) fungicides, has been increasingly applied for the control of tomato gray mold in China. However, very limited information is available on the resistance profile of B. cinerea to tebuconazole. In this study, the baseline sensitivity of B. cinerea to tebuconazole was determined based on 138 isolates from field sites having no history of DMI usage, with a mean EC50 value of 0.29 μg/mL. Another 159 B. cinerea isolates collected in the greenhouse and field from 2011 to 2016 were demonstrated to have a shifted sensitivity to tebuconazole, with a mean EC50 value of 0.66 μg/mL. EC50 values of 10 B. cinerea isolates with reduced sensitivity to tebuconazole were greater than 1.56 μg/mL, and these reduced-sensitive isolates had a fitness penalty in sporulation and conidial germination, but showed similar mycelial growth rate and pathogenicity with those of the sensitive isolates. Positive cross-resistance was observed only between tebuconazole and the other two DMIs difenoconazole and prochloraz, but not between tebuconazole and the non-DMIs iprodione, procymidone, or fludioxonil. In reduced-sensitive isolates, no amino acid variation was found in the BcCYP51 protein. When exposed to tebuconazole, the expression level of BcCYP51 increased in these reduced-sensitive B. cinerea isolates as compared to sensitive ones, thus contributing to the reduced sensitivity of the pathogen to tebuconazole. Additionally, the nucleotide mutation observed in the 1200 bp upstream region of BcCYP51 had no correlation with the development of fungicide resistance in B. cinerea isolates. These findings will be helpful for the understanding of DMI resistance mechnism in B. cinerea.

Abstract Neonicotinoid insecticides are used for preventing insects from transmitting plant viruses. This group of chemicals are easily taken up by plants and translocated to different tissues and are applicable for soil treatment in sustained-release tablets, which greatly reduces environmental contamination compared to foliar spray. The goal of this study was to examine the efficacy of thiamethoxam, acetamiprid and nitenpyram in tablet formulation against whitefly, Bemisia tabaci, the vector of tomato chlorosis virus (ToCV) under laboratory and greenhouse conditions. Effective 50% lethal concentration (LC50) of thiamethoxam, acetamiprid, and nitenpyram on B. tabaci were 2.18, 0.46 and 0.18 μg/g plant tissue, respectively. In vitro test showed that tablets of 10 mg nitenpyram applied in seedbed with thiamethoxam transplanting treatment showed 80.9% control of B. tabaci on tomato and 75.5% of ToCV after 32 days of transplanting. Therefore, neonicotinoid tablets were an effective strategy of soil treatment in controlling viruliferous insects and ToCV with reduced environmental contamination.

Patched (Ptc) and Patched-related (Ptr) proteins containing sterol-sensing domains (SSD) and Patched domains are highly conserved in eukaryotes for lipid transport and metabolism. Four proteins containing predicted SSD and Patched domains were simultaneously found by searching the Phytophthora sojae genome database, and one of them was identified as a Patched-like (PTL) protein. Here, we investigated the biological function of PsPTL. The expression level of PsPTL was higher during mycelial and sporulation stages, compared to zoospore (ZO), cyst, and germinated-cyst stages, without significant change during infection. However, deletion of PsPTL using CRISPR/Cas9 had no significant effect on the growth, development, or virulence of P. sojae. Further investigations showed that PsPTL is not essential for P. sojae to cope with external stresses such as temperature, pH, oxidative and osmotic pressure. In addition, this gene did not appear to play an essential role in P. sojae’s response to exogenous sterols. The transcript levels of the other three proteins containing predicted SSD and Patched domains were also not significantly upregulated in PsPTL deletion transformants. Our studies demonstrated that PsPTL is not an essential protein for P. sojae under the tested conditions, and more in-depth research is required for revealing the potential functions of PsPTL under special conditions or in other signaling pathways.

Abstract Background The widespread occurrence of fungicide resistance in fungal plant pathogens requires the development of new compounds with different mode(s) of action (MOA) to avoid cross resistance. This will require a rapid method to identify MOAs. Results Here, gas chromatography–mass spectrometry (GC–MS) based metabolic fingerprinting was used to elucidate the MOAs of fungicides. Botrytis cinerea, an important pathogen of vegetables and flowers, can be inhibited by a wide range of chemical fungicides with different MOAs. A sensitive strain of B. cinerea was exposed to EC50 concentrations of 13 fungicides with different known MOAs and one with unknown MOA. The mycelial extracts were analyzed for their “metabolic fingerprint” using GC–MS. A comparison among the GC–MS vector’ profiles of cultures treated with fungicides were performeded. A model based on hierarchical clustering was established which allowed these antifungal compounds to be distinguished and classified coinciding with their MOAs. Thus, metabolic fingerprinting represents a rapid, convenient, and information-rich method for classifying the MOAs of antifungal substances. The biomarkers of fungicide MOAs were also established by an analysis of variance and included succinate for succinate dehydrogenase inhibitors and cystathionine for methionine synthesis inhibitors. Using the metabolic model and the common perturbation of metabolites, the new fungicide SYP-14288 was identified as having the same MOA as fluazinam. Conclusion This study provides a comprehensive database of the metabolic perturbations of B. cinerea induced by diverse MOA inhibitors and highlights the utility of metabolic fingerprinting for defining MOAs, which will assist in the development and optimization of new fungicides.

Transcriptional plasticity enables oomycetes to rapidly adapt to environmental challenges including emerging host resistance. For example, the soybean pathogen Phytophthora sojae can overcome resistance conferred by the host resistance gene Rps1b through natural silencing of its corresponding effector gene, Avr1b-1. With the Phytophthora CRISPR/Cas9 genome editing system, it is possible to generate site-specific knock-out (KO) and knock-in (KI) mutants and to investigate the biological functions of target genes. In this study, the Avr1b-1 gene was deleted from the P. sojae genome using a homology-directed recombination strategy that replaced Avr1b-1 with a gene encoding the fluorescent protein mCherry. As expected, all selected KO transformants gained virulence on Rps1b plants, while infection of plants lacking Rps1b was not compromised. When a sgRNA-resistant version of Avr1b-1 was reintroduced into the Avr1b-1 locus of an Avr1b KO transformant, KI transformants with a well-transcribed Avr1b-1 gene were unable to infect Rps1b-containing soybeans. However, loss of expression of the incoming Avr1b-1 gene was frequently observed in KI transformants, which resulted in these transformants readily infecting Rps1b soybeans. A similar variability in the expression levels of the incoming gene was observed with AVI- or mCherry-tagged Avr1b-1 constructs. Our results suggest that Avr1b-1 may be unusually susceptible to transcriptional variation.

The fungicide SYP-14288 has a high efficiency, low toxicity, and broad spectrum in inhibiting both fungi and oomycetes, but its mode of action (MoA) remains unclear on inhibiting fungi. In this study, the MoA was determined by analyzing the metabolism and respiratory activities of Rhizoctonia solani treated by SYP-14288. Wild-type strains and SYP-14288-resistant mutants of R. solani were incubated on potato dextrose agar amended with either SYP-14288 or one of select fungicides acting on fungal respiration, including complex I, II, and III inhibitors; uncouplers; and ATP synthase inhibitors. Mycelial growth was measured under fungicides treatments. ATP content was determined using an ATP assay kit, membrane potential of mitochondria was detected with the JC-1 kit, and respiratory rate was calculated based on the measurement of oxygen consumption of R. solani. A model of metabolic fingerprinting cluster was established to separate oxidation inhibitors and phosphorylation inhibitors. All the results together displayed a clear discrimination between oxidation inhibitors and phosphorylation inhibitors, and the latter inhibited ATP synthase production having or uncoupling activities. Based on the model, SYP-14288 was placed in phosphorylation inhibitor group, because it significantly reduced ATP content and membrane potential of mitochondria while increasing respiratory rate in R. solani. Therefore, the MoA of SYP-14288 on R. solani was confirmed to involve phosphorylation inhibition and possibly uncoupling activity.

Botrytis cinerea is a destructive plant pathogenic ascomycete that causes serious pre- and post-harvest losses worldwide. The novel sterol 14α-demethylase inhibitor (DMI) pyrisoxazole was recently registered for the control of tomato gray mold caused by B. cinerea in China. Baseline sensitivity of 110 B. cinerea isolates collected from nine provinces in China to pyrisoxazole was demonstrated, with a mean EC50 of 0.057 ± 0.029 μg/ml. Eleven stable mutants resistant to pyrisoxazole were generated via UV irradiation (RU-mutants) and spontaneous selection (RS-mutants) of conidia. The efficacy of pyrisoxazole against the resistant mutants was significantly lower than that of the sensitive isolates. Most of the pyrisoxazole- resistant mutants were less fit than the sensitive isolates, with reduced sporulation, conidia germination, sclerotium production, and pathogenicity, which was confirmed by the competitive ability test. Positive cross-resistance was only observed between pyrisoxazole and the DMIs tebuconazole and prochloraz, but not between pyrisoxazole and non-DMIs iprodione, procymidone, diethofencarb, fluazinam, pyrimethanil, or fludioxonil. A two-point mutation, at G476S and K104E in the RU-mutants, and a one point mutation, M231T, in the RS-mutants, were detected in the CYP51 protein of the resistant mutants. When exposed to pyrisoxazole, the induced expression level of CYP51 increased in the resistant isolates as compared to sensitive ones. Molecular docking suggested that G476S and M231T mutations both led to the loss of electrostatic interactions between CYP51 and pyrisoxazole, while no change was found with the K104E mutation. Thus, two point mutations on CYP51 protein combined with induced expression of its target gene appeared to mediate the pyrisoxazole resistance of B cinerea.

Sensitivities of Phytophthora capsici to fluopicolide were investigated in vitro, with results showing that fluopicolide had strong inhibitory activities on each development stage of P. capsici, in particular on the motility of the zoospore. The potential resistance risk for fluopicolide in P. capsici was evaluated. The baseline sensitivities to fluopicolide of 146 isolates obtained from 28 provinces in China were initially determined, and the 50% inhibition of mycelial growth (EC50) distribution was a unimodal curve with a mean of 0.17 μg/ml. A series of fluopicolide-resistant mutants of P. capsici were obtained by fungicide adaptation, and their biological traits were determined. Most of the resistant mutants showed similar favorable fitness in mycelial growth, sporangium and zoospore production, cystospore germination, and pathogenicity compared with their sensitive parents, with few exceptions. Additionally, the cross-resistance result indicated that the sensitivity of fluopicolide did not correlate with other oomycete fungicides, apart from fluopimomide (LH-2010A). These results suggest a moderate to high resistance risk of P. capsici to fluopicolide in China.

Oomycete plant pathogens are difficult to control and routine genetic research is challenging. A major problem is instability of isolates. Here we characterize >600 field and single zoospore isolates of Phytophthora capsici for inheritance of mating type, sensitivity to mefenoxam, chromosome copy number and heterozygous allele frequencies. The A2 mating type was highly unstable with 26% of 241 A2 isolates remaining A2. The A1 mating type was stable. Isolates intermediately resistant to mefenoxam produced fully resistant single-spore progeny. Sensitive isolates remained fully sensitive. Genome re-sequencing of single zoospore isolates revealed extreme aneuploidy; a phenomenon dubbed Dynamic Extreme Aneuploidy (DEA). DEA is characterized by the asexual inheritance of diverse intra-genomic combinations of chromosomal ploidy ranging from 2N to 3N and heterozygous allele frequencies that do not strictly correspond to ploidy. Isolates sectoring on agar media showed dramatically altered heterozygous allele frequencies. DEA can explain the rapid increase of advantageous alleles (e.g. drug resistance), mating type switches and copy neutral loss of heterozygosity (LOH). Although the mechanisms driving DEA are unknown, it can play an important role in adaptation and evolution and seriously hinders all aspects of P. capsici research.

The Hippo pathway regulates organ size, regeneration, and cell growth by controlling the stability of the transcription factor, YAP (Yorkie in Drosophila). When there is tissue damage, YAP is activated allowing the restoration of homeostatic tissue size. The exact signals by which YAP is activated are still not fully understood, but its activation is known to affect both cell size and cell number. Here we used cultured cells to examine the coordinated regulation of cell size and number under the control of YAP. Our experiments in isogenic HEK293 cells reveal that YAP can affect cell size and number by independent circuits. Some of these effects are cell autonomous, such as proliferation, while others are mediated by secreted signals. In particular CYR61, a known secreted YAP target, is a non-cell autonomous mediator of cell survival, while another unidentified secreted factor controls cell size.

Phytophthora, a genus of oomycetes, contains many devastating plant pathogens, which cause substantial economic losses worldwide. Recently, CRISPR/Cas9-based genome editing tool was introduced into Phytophthora to delineate the functionality of individual genes. The available selection markers for Phytophthora transformation, however, are limited, which can restrain transgenic manipulation in some cases. We hypothesized that PcMuORP1, an endogenous fungicide resistance gene from P. capsici that confers resistance to the fungicide oxathiapiprolin via an altered target site in the ORP1 protein, could be used as an alternative marker. To test this hypothesis, the gene PcMuORP1 was introduced into the CRISPR/Cas9 system and complementation of a deleted gene in P. capsici was achieved using it as a selection marker. All of the oxathiapiprolin-resistant transformants were confirmed to contain the marker gene, indicating that the positive screening rate was 100%. The novel selection marker could also be used in other representative Phytophthora species including P. sojae and P. litchii, also with 100% positive screening rate. Furthermore, comparative studies indicated that use of PcMuORP1 resulted in a much higher efficiency of screening compared to the conventional selection marker NPT II, especially in P. capsici. Successive subculture and asexual reproduction in the absence of selective pressure were found to result in the loss of the selection marker from the transformants, which indicates that the PcMuORP1 gene would have little long term influence on the fitness of transformants and could be reused as the selection marker in subsequent projects. Thus, we have created an alternative selection marker for Phytophthora transformation by using a fungicide resistance gene, which would accelerate functional studies of genes in these species.

Point mutation G143A in the cytochrome b (Cyt b) protein commonly confers resistance to quinone outside inhibitor (QoI) fungicides in phytopathogenic fungi, including Corynespora cassiicola, which causes cucumber target spot disease. However, the effect of G143A on the binding between the QoI fungicide and the Cyt b protein, and the use of LAMP (loop-mediated isothermal amplification) to detect this point mutation had not been reported previously in C. cassiicola. In this study, the sensitivity of 131 C. cassiicola isolates—collected from Shandong province, China in 2019 and 2020—to pyraclostrobin was determined. The EC50 values ranged from 1.67 to 8.82 μg/mL, and sequencing results showed that all C. cassiicola isolates contained the G143A mutation. Molecular docking results suggested that G143A significantly alters the affinity of pyraclostrobin to the Cyt b protein. Following development of three LAMP primer pairs, the best reaction condition for LAMP analysis was 65 °C for 60 min, and the detection limit was 0.01 ng/μL of DNA containing the point mutation. In conclusion, the G143A mutation conferring pyraclostrobin resistance is widespread in C. cassiicola from Shandong province, and the LAMP method can be used to monitor QoI resistance in C. cassiicola caused by the G143A mutation in the field.

Proteins that contain the FYVE zinc-finger domain are recruited to PtdIns3P-containing membranes, participating in numerous biological processes such as membrane trafficking, cytoskeletal regulation, and receptor signaling. However, the genome-wide distribution, evolution, and biological functions of FYVE-containing proteins are rarely reported for oomycetes. By genome mining of Phytophthora sojae, two proteins (PsFP1 and PsFP2) with a combination of the FYVE domain and the PX domain (a major phosphoinositide binding module) were found. To clarify the functions of PsFP1 and PsFP2, the CRISPR/Cas9-mediated gene replacement system was used to knock out the two genes respectively. Only heterozygous deletion mutants of PsFP1 were recovered, and the expression level of PsFP1 in the heterozygous knockout transformants was significantly down-regulated. These PsFP1 mutants showed a decrease in mycelial growth and pathogenicity and were more sensitive to hydrogen peroxide. These phenotypes were recovered to the level of wild-type by overexpression PsFP1 gene in the PsFP1 heterozygous knockout transformant. In contrast, deletion of PsFP2 had no significant effect on vegetative growth, asexual and sexual reproduction, pathogenicity, or oxidative stress sensitivity. PsFP1 was primarily localized in vesicle-like structures and both the FYVE and PX domains are important for its localization. Overall, our results indicate that PsFP1 plays an important role in the vegetative growth and virulence of P. sojae.

The probenazole fungicide is used for controlling rice blast (Magnaporthe grisea) primarily by inducing disease resistance of the plant. To investigate the mechanism of induced plant defense, rice seedlings were treated with probenazole at 15 days post emergence, and non-treated plants were used for the control. The plants were infected with M. grisea 5 days after chemical treatment and incubated in a greenhouse. After 7 days, rice seedlings were sampled. The metabolome of rice seedlings was chemically extracted and analyzed using gas chromatography and mass spectrum (GC-MS). The GC-MS data were processed using analysis of variance (ANOVA), principal component analysis (PCA) and metabolic pathway elucidation. Results showed that probenazole application significantly affected the metabolic profile of rice seedlings, and the effect was proportionally leveraged with the increase of probenazole concentration. Probenazole resulted in a change of 54 metabolites. Salicylic acid, γ-aminobutyrate, shikimate and several other primary metabolites related to plant resistance were significantly up-regulated and some metabolites such as phenylalanine, valine and proline were down-regulated in probenazole-treated seedlings. These results revealed a metabolic pathway of rice seedlings induced by probenazole treatment regarding the resistance to M. grisea infection.

Phytophthora capsica is a destructive oomycete plant pathogen that causes huge losses to crop production worldwide. However, the novel fungicide SYP-14288 has shown excellent activity against various stages of the oomycete life cycle as well against fungal plant pathogens. The current study utilized isobaric tags for relative and absolute quantitation technology to generate proteome profiles of P. capsici in the presence or absence of SYP-14288 in order to gain a greater understanding of the SYP-14288 mode of action. A total of 1,443 individual proteins were identified during the investigation, of which 599 were considered to have significantly altered expression. Further investigation using Cluster of Orthologous Groups of proteins analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated most of the proteins with altered expression were associated with carbohydrate metabolism, energy metabolism and their downstream biological functions, especially with regard to oxidoreductase activity and subsequent adenosine triphosphate (ATP) production associated pathways. Quantitative expression analysis using qRT-PCR validated the proteomic data. These results seem to indicate that SYP-14288 treatment caused a shift in energy metabolism that resulted in the activation of compensatory mechanisms affecting carbohydrate and lipid metabolism. The study also found evidence that the up-regulation of transmembrane transporters and proteins associated with stress response might also be coopted to compensate for the disrupted proton gradient and other downstream effects. Taken together these results provide strong evidence that SYP-14288 has a similar mode of action to the oxidative phosphorylation uncoupler Fluazinam but further investigation, including molecular studies, is required to completely characterize the SYP-14288 mode of action in P. capsici. However, the proteomic data collected in the current study does provide important insight into the overall effect of SYP-14288 in P. capsici, which could be useful for the registration and application of this novel fungicide.

In plant-pathogen interaction systems, plant metabolism is usually agitated in the early stages of infection and much before visible symptoms appear. To identify the latent infection of strawberry by Botrytis cinerea by metabolome profiling, a metabolomics method based on gas chromatography and mass spectrometry was applied to identify the affected metabolites and discriminate diseased plants from healthy ones. An orthogonal partial least squares (OPLS) score plot showed that the metabolic profiling well separated B. cinerea -infected strawberry plants at 2, 5, and 7 days after infection from non-infected healthy plants. Combined analysis of variance (ANOVA) and OPLS analysis revealed candidate biomarkers of plant resistance and of infection and expansion of the pathogen in the plants. Among them, hexadecanoic acid, octadecanoic acid, sucrose, β-lyxopyranose, melibiose, and 1,1,4a-Trimethyl-5,6-dimethylenedecahydronaphthalene were closely related to the early stage of disease development when symptoms were not visible. A discrimination method that could distinguish Botrytis gray mold diseased strawberry plants from healthy ones was established based on the partial least squares discriminant analysis (PLS-DA) model with a correct recognition accuracy of 100%. This research offers a good application of metabolome profiling for early diagnosis of plant disease and interaction mechanism exploration.

Botrytis cinerea is a destructive necrotrophic pathogen that can infect many plant species. The control of gray mold mainly relies on the application of fungicides, and the fungicide fludioxonil is widely used in China. However, the field fungicide resistance of B. cinerea to this compound is largely unknown. In this study, B. cinerea isolates were collected from different districts of Shanghai province in 2015–2017, and their sensitivity to fludioxonil was determined. A total of 65 out of 187 field isolates (34.76%) were found to be resistant to fludioxonil, with 36 (19.25%) showing high resistance and 29 (15.51%) showing moderate resistance. Most of these resistant isolates also showed resistance to iprodione, and some developed resistance to fungicides of other modes of action. AtrB gene expression, an indicator of MDR1 and MDR1h phenotypes, was not dramatically increased in the tested resistant isolates. Biological characteristics and osmotic sensitivity investigations showed that the fitness of resistant isolates was lower than that of sensitive ones. To investigate the molecular resistance mechanisms of B. cinerea to fludioxonil, the Bos1 amino acid sequences were compared between resistant and sensitive isolates. Resistant isolates revealed either no amino acid variations or the mutations I365S, I365N, Q369P/N373S, and N373S.

Oxathiapiprolin was developed as a specific plant pathogenic oomycete inhibitor, previously shown to have highly curative and protective activities against the pepper Phytophthora blight disease under field and greenhouse tests. Therefore, it was hypothesized that oxathiapiprolin might potentially activate the plant disease resistance against pathogen infections. This study investigated the potential and related mechanism of oxathiapiprolin to activate the plant disease resistance using the bacterium Pseudomonas syringae pv tomato (Pst) and plant Arabidopsis interaction as the targeted system. Our results showed that oxathiapiprolin could activate the plant disease resistance against Pst DC3000, a non-target pathogen of oxathiapiprolin, in Arabidopsis, tobacco, and tomato plants. Our results also showed the enhanced callose deposition and H2O2 accumulation in the oxathiapiprolin-treated Arabidopsis under the induction of flg22 as the pathogen-associated molecular pattern (PAMP) treatment. Furthermore, increased levels of free salicylic acid (SA) and jasmonic acid (JA) were detected in the oxathiapiprolin-treated Arabidopsis plants compared to the mock-treated ones under the challenge of Pst DC3000. Besides, the gene expression results confirmed that at 24 h after the infiltration with Pst DC3000, the oxathiapiprolin-treated Arabidopsis plants had upregulated expression levels of the respiratory burst oxidase homolog D (RBOHD), JA-responsive gene (PDF1.2), and SA-responsive genes (PR1, PR2, and PR5) compared to the control. Taken together, oxathiapiprolin is identified as a novel chemical inducer which activates the plant disease resistance against Pst DC3000 by enhancing the callose deposition, H2O2 accumulation, and hormone SA and JA production.

The genus Colletotrichum contains a wide variety of important plant pathogens, and Colletotrichum truncatum is one of the most prevalent species of Colletotrichum on chili in China. Demethylation-inhibitor fungicides (DMIs) are currently registered chemical agents for the management of the anthracnose disease caused by Colletotrichum spp. To assess the risk for DMI resistance development, 112 C. truncatum isolates were collected from infected pepper in 13 regions of China. The sensitivity of C. truncatum isolates to five DMI fungicides was determined based on mycelial growth inhibition assay. C. truncatum was sensitive to prochloraz, epoxiconazole, and difenoconazole, but not to tebuconazole or myclobutanil. Baseline sensitivity using the 112 C. truncatum isolates was established for the first three effective DMIs. Prochloraz, epoxiconazole, and difenoconazole EC50 values were 0.053 ± 0.023, 1.956 ± 0.815, and 1.027 ± 0.644 μg/ml, respectively. Eleven stable DMI-resistant mutants all exhibited lower fitness levels than their wild-type parents, suggesting a low risk of DMI resistance in C. truncatum. By inducing gene expression, CtCYP51 expression increased slightly in the resistant mutants as compared to wild-types when exposed to DMI fungicides and thus contributed at least partially to resistance. Molecular docking with CYP51 structure models was used to explain differential sensitivity of the DMI fungicides in C. truncatum. Our results suggest that the M376L/H373N mutations in CYP51 changed the conformation of DMIs in the binding pocket. These changes prevented the formation of the Fe – N coordinate bond between the heme iron active site and tebuconazole or myclobutanil, and apparently contributed to tebuconazole and myclobutanil insensitivity of C. truncatum.

Cell fate decisions are critical for life, yet little is known about how their reliability is achieved when signals are noisy and fluctuating with time. In this study, we show that in budding yeast, the decision of cell cycle commitment (Start) is determined by the time integration of its triggering signal Cln3. We further identify the Start repressor, Whi5, as the integrator. The instantaneous kinase activity of Cln3-Cdk1 is recorded over time on the phosphorylated Whi5, and the decision is made only when phosphorylated Whi5 reaches a threshold. Cells adjust the threshold by modulating Whi5 concentration in different nutrient conditions to coordinate growth and division. Our work shows that the strategy of signal integration, which was previously found in decision-making behaviors of animals, is adopted at the cellular level to reduce noise and minimize uncertainty.

This study characterized isolates of P. capsici that had developed a novel mechanism of resistance to zoxamide, which altered the minimum inhibition concentration (MIC) but not the EC50. Molecular analysis revealed that the β-tubulin gene of the resistant isolates contained no mutations and was expressed at the same level as in zoxamide-sensitive isolates. This suggested that P. capsici had developed a novel non-target-site-based resistance to zoxamide. Analysis of the segregation ratio of zoxamide-resistance in the sexual progeny of the sensitive isolates PCAS1 and PCAS2 indicated that the resistance to zoxamide was controlled by one or more recessive nuclear genes. Furthermore, the segregation of resistance in the F1, F2, and BC1 progeny was in accordance with the theoretical ratios of the χ(2) test (P>0.05), which suggested that the resistance to zoxamide was controlled by two recessive genes, and that resistance to zoxamide occurred when at least one pair of these alleles was homozygous. This implies that the risk of zoxamide-resistance in P. capsici is low to moderate. Nevertheless this potential for resistance should be monitored closely, especially if two compatible mating types co-exist in the same field.

Pyrimorph is a novel fungicide with high activity against the plant pathogen Phytophthora capsici. We investigated the risk that P. capsici can develop resistance to pyrimorph. The baseline sensitivities of 226 P. capsici isolates, tested by mycelial growth inhibition, showed a unimodal distribution with a mean EC(50) value of 1.4261 (± 0.4002) µg/ml. Twelve pyrimorph-resistant mutants were obtained by repeated exposure to pyrimorph in vitro with a frequency of approximately 1 × 10(-4). The resistance factors of the mutants ranged from 10.67 to 56.02. Pyrimorph resistance of the mutants was stable after 10 transfers on pyrimorph-free medium. Fitness in sporulation, cystospore germination, and pathogenicity in the pyrimorph-resistant mutants was similar to or less than that in the parental wild-type isolates. On detached pepper leaves and pepper plants treated with the recommended maximum dose of pyrimorph, however, virulence was greater for mutants with a high level of pyrimorph resistance than for the wild type. The results suggest that the risk of P. capsici developing resistance to pyrimorph is low to moderate. Among mutants with a high level of pyrimorph resistance, EC(50) values for pyrimorph and CAA fungicides flumorph, dimethomorph, and mandipropamid were positively correlated. This indicated that point mutations in cellulose synthase 3 (CesA3) may confer resistance to pyrimorph. Comparison of CesA3 in isolates with a high level of pyrimorph resistance and parental isolates showed that an amino acid change from glutamine to lysine at position 1077 resulted in stable, high resistance in the mutants. Based on the point mutations, an allele-specific PCR method was developed to detect pyrimorph resistance in P. capsici populations.

Phytophthora capsici causes significant loss to pepper (Capsicum annum) in China and our goal was to develop single nucleotide polymorphism (SNP) markers for P. capsici and characterize genetic diversity nationwide. Eighteen isolates of P. capsici from locations worldwide were re-sequenced and candidate nuclear and mitochondrial SNPs identified. From 2006 to 2012, 276 isolates of P. capsici were recovered from 136 locations in 27 provinces and genotyped using 45 nuclear and 2 mitochondrial SNPs. There were two main mitochondrial haplotypes and 95 multi-locus genotypes (MLGs) identified. Genetic diversity was geographically structured with a high level of genotypic diversity in the north and on Hainan Island in the south, suggesting outcrossing contributes to diversity in these areas. The remaining areas of China are dominated by four clonal lineages that share mitochondrial haplotypes, are almost exclusively the A1 or A2 mating type and appear to exhibit extensive diversity based on loss of heterozygosity (LOH). Analysis of SNPs directly from infected peppers confirmed LOH in field populations. One clonal lineage is dominant throughout much of the country. The overall implications for long-lived genetically diverse clonal lineages amidst a widely dispersed sexual population are discussed.

The risk that the plant pathogen Phytophthora melonis develops resistance to carboxylic acid amide (CAA) fungicides was determined by measuring baseline sensitivities of field isolates, generating resistant mutants, and measuring the fitness of the resistant mutants. The baseline sensitivities of 80 isolates to flumorph, dimethomorph and iprovalicarb were described by unimodal curves, with mean EC(50) values of 0.986 (±0.245), 0.284 (±0.060) and 0.327 (±0.068) µg/ml, respectively. Seven isolates with different genetic background (as indicated by RAPD markers) were selected to generate CAA-resistance. Fifty-five resistant mutants were obtained from three out of seven isolates by spontaneous selection and UV-mutagenesis with frequencies of 1×10(-7) and 1×10(-6), respectively. CAA-resistance was stable for all mutants. The resistance factors of these mutants ranged from 7 to 601. The compound fitness index (CFI = mycelial growth × zoospore production × pathogenicity) was often lower for the CAA-resistant isolates than for wild-type isolates, suggesting that the risk of P. melonis developing resistance to CAA fungicides is low to moderate. Among the CAA-resistant isolates, a negative correlation between EC(50) values was found for iprovalicarb vs. flumorph and for iprovalicarb vs. dimethomorph. Comparison of the full-length cellulose synthase 3 (CesA3) between wild-type and CAA-resistant isolates revealed only one point mutation at codon position 1109: a valine residue (codon GTG in wild-type isolates) was converted to leucine (codon CTG in resistant mutants). This represents a novel point mutation with respect to mutations in CesA3 conferring resistance to CAA fungicides. Based on this mutation, an efficient allelic-specific PCR (AS-PCR) method was developed for rapid detection of CAA-resistance in P. melonis populations.

Design and synthesis of basic functional circuits are the fundamental tasks of synthetic biologists. Before it is possible to engineer higher‐order genetic networks that can perform complex functions, a toolkit of basic devices must be developed. Among those devices, sequential logic circuits are expected to be the foundation of the genetic information‐processing systems. In this study, we report the design and construction of a genetic sequential logic circuit in Escherichia coli. It can generate different outputs in response to the same input signal on the basis of its internal state, and ‘memorize’ the output. The circuit is composed of two parts: (1) a bistable switch memory module and (2) a double‐repressed promoter NOR gate module. The two modules were individually rationally designed, and they were coupled together by fine‐tuning the interconnecting parts through directed evolution. After fine‐tuning, the circuit could be repeatedly, alternatively triggered by the same input signal; it functions as a push‐on push‐off switch.