Fungicide Sensitivity of Sclerotinia sclerotiorum from U.S. Soybean and Dry Bean, Compared to Different Regions and Climates.

Fungicide use is integral to reduce yield loss from Sclerotinia sclerotiorum on dry bean and soybean. Increasing fungicide use against this fungus may lead to resistance to the most common fungicides. Resistance has been reported in Brazil (Glycine max) and China (Brassica napus subsp. napus), however, few studies have investigated fungicide sensitivity of S. sclerotiorum in the United States. This work was conducted to determine if there was a difference in fungicide sensitivity of S. sclerotiorum isolates in the United States from: (i) dry bean versus soybean and (ii) fields with different frequencies of fungicide application. We further hypothesized that isolates with fungicide applications of a single active ingredient from tropical Brazil and subtropical Mexico were less sensitive than temperate U.S. isolates due to different management practices and climates. The EC50(D) fungicide sensitivity of 512 S. sclerotiorum isolates from the United States (443), Brazil (36), and Mexico (33) was determined using a discriminatory concentration (DC) previously identified for tetraconazole (2.0 ppm; EC50(D) range of 0.197 to 2.27 ppm), boscalid (0.2; 0.042 to 0.222), picoxystrobin (0.01; 0.006 to 0.027), and thiophanate-methyl, which had a qualitative DC of 10 ppm. Among the 10 least sensitive isolates to boscalid and picoxystrobin, 2 presented mutations known to confer resistance in the SdhB (qualitative) and SdhC (quantitative) genes; however, no strong resistance was found. This study established novel DCs that can be used for further resistance monitoring and baseline sensitivity of S. sclerotiorum to tetraconazole worldwide plus baseline sensitivity to boscalid in the United States.

The conservation of IAP-like proteins in fungi, and their potential role in fungal programmed cell death.

Programmed cell death (PCD) is a tightly regulated process which is required for survival and proper development of all cellular life. Despite this ubiquity, the precise molecular underpinnings of PCD have been primarily characterized in animals. Attempts to expand our understanding of this process in fungi have proven difficult as core regulators of animal PCD are apparently absent in fungal genomes, with the notable exception of a class of proteins referred to as inhibitors of apoptosis proteins (IAPs). These proteins are characterized by the conservation of a distinct Baculovirus IAP Repeat (BIR) domain and animal IAPs are known to regulate a number of processes, including cellular death, development, organogenesis, immune system maturation, host-pathogen interactions and more. IAP homologs are broadly conserved throughout the fungal kingdom, but our understanding of both their mechanism and role in fungal development/virulence is still unclear. In this review, we provide a broad and comparative overview of IAP function across taxa, with a particular focus on fungal processes regulated by IAPs. Furthermore, their putative modes of action in the absence of canonical interactors will be discussed.

Identification of Soybean (Glycine max) Check Lines for Evaluating Genetic Resistance to Sclerotinia Stem Rot.

Soybean production in the upper midwestern United States is affected by Sclerotinia stem rot (SSR) caused by the fungal pathogen Sclerotinia sclerotiorum. Genetic resistance is an important management strategy for this disease; however, assessing genetic resistance to S. sclerotiorum is challenging because a standardized method of examining resistance across genotypes is lacking. Using a panel of nine diverse S. sclerotiorum isolates, four soybean lines were assessed for reproducible responses to S. sclerotiorum infection. Significant differences in SSR severity were found across isolates (P < 0.01) and soybean lines (P < 0.01), including one susceptible, two moderately resistant, and one highly resistant line. These four validated lines were used to screen 11 other soybean genotypes to evaluate their resistance levels, and significant differences were found across genotypes (P < 0.01). Among these 11 genotypes, five commercial and public cultivars displayed high resistance and were assessed during field studies across the upper midwestern United States growing region to determine their response to SSR and yield. These five cultivars resulted in low disease levels (P < 0.01) in the field that were consistent with greenhouse experiment results. The yields were significantly different in fields with disease present (P < 0.01) and disease absent (P < 0.01), and the order of cultivar performance was consistent between environments where disease was present or absent, suggesting that resistance prevented yield loss to disease. This study suggests that the use of a soybean check panel can accurately assess SSR resistance in soybean germplasm and aid in breeding and commercial soybean development.

Gene regulation of Sclerotinia sclerotiorum during infection of Glycine max: on the road to pathogenesis.

BACKGROUND: Sclerotinia sclerotiorum is a broad-host range necrotrophic pathogen which is the causative agent of Sclerotinia stem rot (SSR), and a major disease of soybean (Glycine max). A time course transcriptomic analysis was performed in both compatible and incompatible soybean lines to identify pathogenicity and developmental factors utilized by S. sclerotiorum to achieve pathogenic success. RESULTS: A comparison of genes expressed during early infection identified the potential importance of toxin efflux and nitrogen metabolism during the early stages of disease establishment. The later stages of infection were characterized by an apparent shift to survival structure formation. Analysis of genes highly upregulated in-planta revealed a temporal regulation of hydrolytic and detoxification enzymes, putative secreted effectors, and secondary metabolite synthesis genes. Redox regulation also appears to play a key role during the course of infection, as suggested by the high expression of genes involved in reactive oxygen species production and scavenging. Finally, distinct differences in early gene expression were noted based on the comparison of S. sclerotiorum infection of resistant and susceptible soybean lines. CONCLUSIONS: Although many potential virulence factors have been noted in the S. sclerotiorum pathosystem, this study serves to highlight soybean specific processes most likely to be critical in successful infection. Functional studies of genes identified in this work are needed to confirm their importance to disease development, and may constitute valuable targets of RNAi approaches to improve resistance to SSR.

Resistance against Sclerotinia sclerotiorum in soybean involves a reprogramming of the phenylpropanoid pathway and up-regulation of antifungal activity targeting ergosterol biosynthesis.

Sclerotinia sclerotiorum, a predominately necrotrophic fungal pathogen with a broad host range, causes a significant yield-limiting disease of soybean called Sclerotinia stem rot. Resistance mechanisms against this pathogen in soybean are poorly understood, thus hindering the commercial deployment of resistant varieties. We used a multiomic approach utilizing RNA-sequencing, gas chromatography-mass spectrometry-based metabolomics and chemical genomics in yeast to decipher the molecular mechanisms governing resistance to S. sclerotiorum in soybean. Transcripts and metabolites of two soybean recombinant inbred lines, one resistant and one susceptible to S. sclerotiorum were analysed in a time course experiment. The combined results show that resistance to S. sclerotiorum in soybean is associated in part with an early accumulation of JA-Ile ((+)-7-iso-jasmonoyl-L-isoleucine), a bioactive jasmonate, increased ability to scavenge reactive oxygen species, and importantly, a reprogramming of the phenylpropanoid pathway leading to increased antifungal activities. Indeed, we noted that phenylpropanoid pathway intermediates, such as 4-hydroxybenzoate, cinnamic acid, ferulic acid and caffeic acid, were highly accumulated in the resistant line. In vitro assays show that these metabolites and total stem extracts from the resistant line clearly affect S. sclerotiorum growth and development. Using chemical genomics in yeast, we further show that this antifungal activity targets ergosterol biosynthesis in the fungus, by disrupting enzymes involved in lipid and sterol biosynthesis. Overall, our results are consistent with a model where resistance to S. sclerotiorum in soybean coincides with an early recognition of the pathogen, leading to the modulation of the redox capacity of the host and the production of antifungal metabolites.

Aspartyl Protease-Mediated Cleavage of BAG6 Is Necessary for Autophagy and Fungal Resistance in Plants.

The Bcl-2-associated athanogene (BAG) family is an evolutionarily conserved group of cochaperones that modulate numerous cellular processes. Previously we found that Arabidopsis thaliana BAG6 is required for basal immunity against the fungal phytopathogen Botrytis cinerea. However, the mechanisms by which BAG6 controls immunity are obscure. Here, we address this important question by determining the molecular mechanisms responsible for BAG6-mediated basal resistance. We show that Arabidopsis BAG6 is cleaved in vivo in a caspase-1-like-dependent manner and via a combination of pull-downs, mass spectrometry, yeast two-hybrid assays, and chemical genomics, we demonstrate that BAG6 interacts with a C2 GRAM domain protein (BAGP1) and an aspartyl protease (APCB1), both of which are required for BAG6 processing. Furthermore, fluorescence and transmission electron microscopy established that BAG6 cleavage triggers autophagy in the host that coincides with disease resistance. Targeted inactivation of BAGP1 or APCB1 results in the blocking of BAG6 processing and loss of resistance. Mutation of the cleavage site blocks cleavage and inhibits autophagy in plants; disease resistance is also compromised. Taken together, these results identify a mechanism that couples an aspartyl protease with a molecular cochaperone to trigger autophagy and plant defense, providing a key link between fungal recognition and the induction of cell death and resistance.

Meta-Analytic and Economic Approaches for Evaluation of Pesticide Impact on Sclerotinia Stem Rot Control and Soybean Yield in the North Central United States.

As complete host resistance in soybean has not been achieved, Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum continues to be of major economic concern for farmers. Thus, chemical control remains a prevalent disease management strategy. Pesticide evaluations were conducted in Illinois, Iowa, Michigan, Minnesota, New Jersey, and Wisconsin from 2009 to 2016, for a total of 25 site-years (n = 2,057 plot-level data points). These studies were used in network meta-analyses to evaluate the impact of 10 popular pesticide active ingredients, and seven common application timings on SSR control and yield benefit, compared with not treating with a pesticide. Boscalid and picoxystrobin frequently offered the best reductions in disease severity and best yield benefit (P < 0.0001). Pesticide applications (one- or two-spray programs) made during the bloom period provided significant reductions in disease severity index (DIX) (P < 0.0001) and led to significant yield benefits (P = 0.0009). Data from these studies were also used in nonlinear regression analyses to determine the effect of DIX on soybean yield. A three-parameter logistic model was found to best describe soybean yield loss (pseudo-R2 = 0.309). In modern soybean cultivars, yield loss due to SSR does not occur until 20 to 25% DIX, and considerable yield loss (-697 kg ha-1 or -10 bu acre-1) is observed at 68% DIX. Further analyses identified several pesticides and programs that resulted in greater than 60% probability for return on investment under high disease levels.

Selenate sensitivity of a laeA mutant is restored by overexpression of the bZIP protein MetR in Aspergillus fumigatus.

LaeA is a conserved global regulator of secondary metabolism and development in filamentous fungi. Examination of Aspergillus fumigatus transcriptome data of laeA deletion mutants have been fruitful in identifying genes and molecules contributing to the laeA mutant phenotype. One of the genes significantly down regulated in A. fumigatus ΔlaeA is metR, encoding a bZIP DNA binding protein required for sulfur and methionine metabolism in fungi. LaeA and MetR deletion mutants exhibit several similarities including down regulation of sulfur assimilation and methionine metabolism genes and ability to grow on the toxic sulfur analog, sodium selenate. However, unlike ΔmetR, ΔlaeA strains are able to grow on sulfur, sulfite, and cysteine. To examine if any parameter of the ΔlaeA phenotype is due to decreased metR expression, an over-expression allele (OE::metR) was placed in a ΔlaeA background. The OE::metR allele could not significantly restore expression of MetR regulated genes in ΔlaeA but did restore sensitivity to sodium selenate. In A. nidulans a second bZIP protein, MetZ, also regulates sulfur and methionine metabolism genes. However, addition of an OE::metZ construct to the A. fumigatus ΔlaeA OE::metR strain still was unable to rescue the ΔlaeA phenotype to wildtype with regards gliotoxin synthesis and virulence in a zebrafish aspergillosis model.

Plant-derived antifungal agent poacic acid targets ß-1,3-glucan.

A rise in resistance to current antifungals necessitates strategies to identify alternative sources of effective fungicides. We report the discovery of poacic acid, a potent antifungal compound found in lignocellulosic hydrolysates of grasses. Chemical genomics using Saccharomyces cerevisiae showed that loss of cell wall synthesis and maintenance genes conferred increased sensitivity to poacic acid. Morphological analysis revealed that cells treated with poacic acid behaved similarly to cells treated with other cell wall-targeting drugs and mutants with deletions in genes involved in processes related to cell wall biogenesis. Poacic acid causes rapid cell lysis and is synergistic with caspofungin and fluconazole. The cellular target was identified; poacic acid localized to the cell wall and inhibited ß-1,3-glucan synthesis in vivo and in vitro, apparently by directly binding ß-1,3-glucan. Through its activity on the glucan layer, poacic acid inhibits growth of the fungi Sclerotinia sclerotiorum and Alternaria solani as well as the oomycete Phytophthora sojae. A single application of poacic acid to leaves infected with the broad-range fungal pathogen S. sclerotiorum substantially reduced lesion development. The discovery of poacic acid as a natural antifungal agent targeting ß-1,3-glucan highlights the potential side use of products generated in the processing of renewable biomass toward biofuels as a source of valuable bioactive compounds and further clarifies the nature and mechanism of fermentation inhibitors found in lignocellulosic hydrolysates.

Validating Sclerotinia sclerotiorum Apothecial Models to Predict Sclerotinia Stem Rot in Soybean (Glycine max) Fields.

In soybean, Sclerotinia sclerotiorum apothecia are the sources of primary inoculum (ascospores) critical for Sclerotinia stem rot (SSR) development. We recently developed logistic regression models to predict the presence of apothecia in irrigated and nonirrigated soybean fields. In 2017, small-plot trials were established to validate two weather-based models (one for irrigated fields and one for nonirrigated fields) to predict SSR development. Additionally, apothecial scouting and disease monitoring were conducted in 60 commercial fields in three states between 2016 and 2017 to evaluate model accuracy across the growing region. Site-specific air temperature, relative humidity, and wind speed data were obtained through the Integrated Pest Information Platform for Extension and Education (iPiPE) and Dark Sky weather networks. Across all locations, iPiPE-driven model predictions during the soybean flowering period (R1 to R4 growth stages) explained end-of-season disease observations with an accuracy of 81.8% using a probability action threshold of 35%. Dark Sky data, incorporating bias corrections for weather variables, explained end-of-season disease observations with 87.9% accuracy (in 2017 commercial locations in Wisconsin) using a 40% probability threshold. Overall, these validations indicate that these two weather-based apothecial models, using either weather data source, provide disease risk predictions that both reduce unnecessary chemical application and accurately advise applications at critical times.

Weather-Based Models for Assessing the Risk of Sclerotinia sclerotiorum Apothecial Presence in Soybean (Glycine max) Fields.

Sclerotinia stem rot (SSR) epidemics in soybean, caused by Sclerotinia sclerotiorum, are currently responsible for annual yield reductions in the United States of up to 1 million metric tons. In-season disease management is largely dependent on chemical control but its efficiency and cost-effectiveness depends on both the chemistry used and the risk of apothecia formation, germination, and further dispersal of ascospores during susceptible soybean growth stages. Hence, accurate prediction of the S. sclerotiorum apothecial risk during the soybean flowering period could enable farmers to improve in-season SSR management. From 2014 to 2016, apothecial presence or absence was monitored in three irrigated (n = 1,505 plot-level observations) and six nonirrigated (n = 2,361 plot-level observations) field trials located in Iowa (n = 156), Michigan (n = 1,400), and Wisconsin (n = 2,310), for a total of 3,866 plot-level observations. Hourly air temperature, relative humidity, dew point, wind speed, leaf wetness, and rainfall were also monitored continuously, throughout the season, at each location using high-resolution gridded weather data. Logistic regression models were developed for irrigated and nonirrigated conditions using apothecial presence as a binary response variable. Agronomic variables (row width) and weather-related variables (defined as 30-day moving averages, prior to apothecial presence) were tested for their predictive ability. In irrigated soybean fields, apothecial presence was best explained by row width (r = -0.41, P < 0.0001), 30-day moving averages of daily maximum air temperature (r = 0.27, P < 0.0001), and daily maximum relative humidity (r = 0.16, P < 0.05). In nonirrigated fields, apothecial presence was best explained by using moving averages of daily maximum air temperature (r = -0.30, P < 0.0001) and wind speed (r = -0.27, P < 0.0001). These models correctly predicted (overall accuracy of 67 to 70%) apothecial presence during the soybean flowering period for four independent datasets (n = 1,102 plot-level observations or 30 daily mean observations).

Cell death control: the interplay of apoptosis and autophagy in the pathogenicity of Sclerotinia sclerotiorum.

Programmed cell death is characterized by a cascade of tightly controlled events that culminate in the orchestrated death of the cell. In multicellular organisms autophagy and apoptosis are recognized as two principal means by which these genetically determined cell deaths occur. During plant-microbe interactions cell death programs can mediate both resistant and susceptible events. Via oxalic acid (OA), the necrotrophic phytopathogen Sclerotinia sclerotiorum hijacks host pathways and induces cell death in host plant tissue resulting in hallmark apoptotic features in a time and dose dependent manner. OA-deficient mutants are non-pathogenic and trigger a restricted cell death phenotype in the host that unexpectedly exhibits markers associated with the plant hypersensitive response including callose deposition and a pronounced oxidative burst, suggesting the plant can recognize and in this case respond, defensively. The details of this plant directed restrictive cell death associated with OA deficient mutants is the focus of this work. Using a combination of electron and fluorescence microscopy, chemical effectors and reverse genetics, we show that this restricted cell death is autophagic. Inhibition of autophagy rescued the non-pathogenic mutant phenotype. These findings indicate that autophagy is a defense response in this necrotrophic fungus/plant interaction and suggest a novel function associated with OA; namely, the suppression of autophagy. These data suggest that not all cell deaths are equivalent, and though programmed cell death occurs in both situations, the outcome is predicated on who is in control of the cell death machinery. Based on our data, we suggest that it is not cell death per se that dictates the outcome of certain plant-microbe interactions, but the manner by which cell death occurs that is crucial.

A single laccase acts as a key component of environmental sensing in a broad host range fungal pathogen.

Secreted laccases are important enzymes on a broad ecological scale for their role in mediating plant-microbe interactions, but within ascomycete fungi these enzymes have been primarily associated with melanin biosynthesis. In this study, a putatively secreted laccase, Sslac2, was characterized from the broad-host-range plant pathogen Sclerotinia sclerotiorum, which is largely unpigmented and is not dependent on melanogenesis for plant infection. Gene knockouts of Sslac2 demonstrate wide ranging developmental phenotypes and are functionally non-pathogenic. These mutants also displayed indiscriminate growth behaviors and enhanced biomass formation, seemingly as a result of their inability to respond to canonical environmental growth cues, a phenomenon further confirmed through chemical stress, physiological, and transcriptomic analyses. Transmission and scanning electron microscopy demonstrate apparent differences in extracellular matrix structure between WT and mutant strains that likely explain the inability of the mutants to respond to their environment. Targeting Sslac2 using host-induced gene silencing significantly improved resistance to S. sclerotiorum, suggesting that fungal laccases could be a valuable target of disease control. Collectively, we identified a laccase critical to the development and virulence of the broad-host-range pathogen S. sclerotiorum and propose a potentially novel role for fungal laccases in modulating environmental sensing.

Tipping the balance: Sclerotinia sclerotiorum secreted oxalic acid suppresses host defenses by manipulating the host redox environment.

Sclerotinia sclerotiorum is a necrotrophic ascomycete fungus with an extremely broad host range. This pathogen produces the non-specific phytotoxin and key pathogenicity factor, oxalic acid (OA). Our recent work indicated that this fungus and more specifically OA, can induce apoptotic-like programmed cell death (PCD) in plant hosts, this induction of PCD and disease requires generation of reactive oxygen species (ROS) in the host, a process triggered by fungal secreted OA. Conversely, during the initial stages of infection, OA also dampens the plant oxidative burst, an early host response generally associated with plant defense. This scenario presents a challenge regarding the mechanistic details of OA function; as OA both suppresses and induces host ROS during the compatible interaction. In the present study we generated transgenic plants expressing a redox-regulated GFP reporter. Results show that initially, Sclerotinia (via OA) generates a reducing environment in host cells that suppress host defense responses including the oxidative burst and callose deposition, akin to compatible biotrophic pathogens. Once infection is established however, this necrotroph induces the generation of plant ROS leading to PCD of host tissue, the result of which is of direct benefit to the pathogen. In contrast, a non-pathogenic OA-deficient mutant failed to alter host redox status. The mutant produced hypersensitive response-like features following host inoculation, including ROS induction, callose formation, restricted growth and cell death. These results indicate active recognition of the mutant and further point to suppression of defenses by the wild type necrotrophic fungus. Chemical reduction of host cells with dithiothreitol (DTT) or potassium oxalate (KOA) restored the ability of this mutant to cause disease. Thus, Sclerotinia uses a novel strategy involving regulation of host redox status to establish infection. These results address a long-standing issue involving the ability of OA to both inhibit and promote ROS to achieve pathogenic success.

AtBAG7, an Arabidopsis Bcl-2-associated athanogene, resides in the endoplasmic reticulum and is involved in the unfolded protein response.

The Bcl-2-associated athanogene (BAG) family is an evolutionarily conserved, multifunctional group of cochaperones that perform diverse cellular functions ranging from proliferation to growth arrest and cell death in yeast, in mammals, and, as recently observed, in plants. The Arabidopsis genome contains seven homologs of the BAG family, including four with domain organization similar to animal BAGs. In the present study we show that an Arabidopsis BAG, AtBAG7, is a uniquely localized endoplasmic reticulum (ER) BAG that is necessary for the proper maintenance of the unfolded protein response (UPR). AtBAG7 was shown to interact directly in vivo with the molecular chaperone, AtBiP2, by bimolecular fluorescence complementation assays, and the interaction was confirmed by yeast two-hybrid assay. Treatment with an inducer of UPR, tunicamycin, resulted in accelerated cell death of AtBAG7-null mutants. Furthermore, AtBAG7 knockouts were sensitive to known ER stress stimuli, heat and cold. In these knockouts heat sensitivity was reverted successfully to the wild-type phenotype with the addition of the chemical chaperone, tauroursodexycholic acid (TUDCA). Real-time PCR of ER stress proteins indicated that the expression of the heat-shock protein, AtBiP3, is selectively up-regulated in AtBAG7-null mutants upon heat and cold stress. Our results reveal an unexpected diversity of the plant's BAG gene family and suggest that AtBAG7 is an essential component of the UPR during heat and cold tolerance, thus confirming the cytoprotective role of plant BAGs.

A broadly conserved fungal alcohol oxidase (AOX) facilitates fungal invasion of plants.

Alcohol oxidases (AOXs) are ecologically important enzymes that facilitate a number of plant-fungal interactions. Within Ascomycota they are primarily associated with methylotrophy, as a peroxisomal AOX catalysing the conversion of methanol to formaldehyde in methylotrophic yeast. In this study we demonstrate that AOX orthologues are phylogenetically conserved proteins that are common in the genomes of nonmethylotrophic, plant-associating fungi. Additionally, AOX orthologues are highly expressed during infection in a range of diverse pathosystems. To study the role of AOX in plant colonization, AOX knockout mutants were generated in the broad host range pathogen Sclerotinia sclerotiorum. Disease assays in soybean showed that these mutants had a significant virulence defect as evidenced by markedly reduced stem lesions and mortality rates. Chemical genomics suggested that SsAOX may function as an aromatic AOX, and growth assays demonstrated that ΔSsAOX is incapable of properly utilizing plant extract as a nutrient source. Profiling of known aromatic alcohols pointed towards the monolignol coniferyl alcohol (CA) as a possible substrate for SsAOX. As CA and other monolignols are ubiquitous among land plants, the presence of highly conserved AOX orthologues throughout Ascomycota implies that this is a broadly conserved protein used by ascomycete fungi during plant colonization.

The cytoprotective co-chaperone, AtBAG4, supports increased nodulation and seed protein content in chickpea without yield penalty.

Drought and extreme temperatures significantly limit chickpea productivity worldwide. The regulation of plant programmed cell death pathways is emerging as a key component of plant stress responses to maintain homeostasis at the cellular-level and a potential target for crop improvement against environmental stresses. Arabidopsis thaliana Bcl-2 associated athanogene 4 (AtBAG4) is a cytoprotective co-chaperone that is linked to plant responses to environmental stress. Here, we investigate whether exogenous expression of AtBAG4 impacts nodulation and nitrogen fixation. Transgenic chickpea lines expressing AtBAG4 are more drought tolerant and produce higher yields under drought stress. Furthermore, AtBAG4 expression supports higher nodulation, photosynthetic levels, nitrogen fixation and seed nitrogen content under well-watered conditions when the plants were inoculated with Mesorhizobium ciceri. Together, our findings illustrate the potential use of cytoprotective chaperones to improve crop performance at least in the greenhouse in future uncertain climates with little to no risk to yield under well-watered and water-deficient conditions.

High-throughput platform for yeast morphological profiling predicts the targets of bioactive compounds.

Morphological profiling is an omics-based approach for predicting intracellular targets of chemical compounds in which the dose-dependent morphological changes induced by the compound are systematically compared to the morphological changes in gene-deleted cells. In this study, we developed a reliable high-throughput (HT) platform for yeast morphological profiling using drug-hypersensitive strains to minimize compound use, HT microscopy to speed up data generation and analysis, and a generalized linear model to predict targets with high reliability. We first conducted a proof-of-concept study using six compounds with known targets: bortezomib, hydroxyurea, methyl methanesulfonate, benomyl, tunicamycin, and echinocandin B. Then we applied our platform to predict the mechanism of action of a novel diferulate-derived compound, poacidiene. Morphological profiling of poacidiene implied that it affects the DNA damage response, which genetic analysis confirmed. Furthermore, we found that poacidiene inhibits the growth of phytopathogenic fungi, implying applications as an effective antifungal agent. Thus, our platform is a new whole-cell target prediction tool for drug discovery.

Bacterial effector HopF2 suppresses arabidopsis innate immunity at the plasma membrane.

Many bacterial pathogens inject a cocktail of effector proteins into host cells through type III secretion systems. These effectors act in concert to modulate host physiology and immune signaling, thereby promoting pathogenicity. In a search for additional Pseudomonas syringae effectors in suppressing plant innate immunity triggered by pathogen or microbe-associated molecular patterns (PAMPs or MAMPs), we identified P. syringae tomato DC3000 effector HopF2 as a potent suppressor of early immune-response gene transcription and mitogen-activated protein kinase (MAPK) signaling activated by multiple MAMPs, including bacterial flagellin, elongation factor Tu, peptidoglycan, lipopolysaccharide and HrpZ1 harpin, and fungal chitin. The conserved surface-exposed residues of HopF2 are essential for its MAMP suppression activity. HopF2 is targeted to the plant plasma membrane through a putative myristoylation site, and the membrane association appears to be required for its MAMP-suppression function. Expression of HopF2 in plants potently diminished the flagellin-induced phosphorylation of BIK1, a plasma membrane-associated cytoplasmic kinase that is rapidly phosphorylated within one minute upon flagellin perception. Thus, HopF2 likely intercepts MAMP signaling at the plasma membrane immediately of signal perception. Consistent with the potent suppression function of multiple MAMP signaling, expression of HopF2 in transgenic plants compromised plant nonhost immunity to bacteria P. syringae pv. Phaseolicola and plant immunity to the necrotrophic fungal pathogen Botrytis cinerea.

Identification and characterization of Sclerotinia sclerotiorum NADPH oxidases.

Numerous studies have shown both the detrimental and beneficial effects of reactive oxygen species (ROS) in animals, plants, and fungi. These organisms utilize controlled generation of ROS for signaling, pathogenicity, and development. Here, we show that ROS are essential for the pathogenic development of Sclerotinia sclerotiorum, an economically important fungal pathogen with a broad host range. Based on the organism's completed genome sequence, we identified two S. sclerotiorum NADPH oxidases (SsNox1 and SsNox2), which presumably are involved in ROS generation. RNA interference (RNAi) was used to examine the function of SsNox1 and SsNox2. Silencing of SsNox1 expression indicated a central role for this enzyme in both virulence and pathogenic (sclerotial) development, while inactivation of the SsNox2 gene resulted in limited sclerotial development, but the organism remained fully pathogenic. ΔSsnox1 strains had reduced ROS levels, were unable to develop sclerotia, and unexpectedly correlated with significantly reduced oxalate production. These results are in accordance with previous observations indicating that fungal NADPH oxidases are required for pathogenic development and are consistent with the importance of ROS regulation in the successful pathogenesis of S. sclerotiorum.