Mehdi Kabbage

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.

Pathogenic attributes of Sclerotinia sclerotiorum: switching from a biotrophic to necrotrophic lifestyle.

Plants and fungi have had many years of friendly and not-so friendly competition for resources and quality of life. As a result, diverse pathosystems evolved numerous strategies, coupled with the emergence of multifaceted pathogenic and saprophytic lifestyles. We discuss fungal lifestyle classifications and how the views associated with certain fungal pathogens, particularly necrotophs, are changing as we learn more about the complexities of their interactions with a given host plant. We discuss the physiological events leading to the transition from biotrophy to necrotrophy in hemi-biotrophs, and conclude that both the control of plant immune responses and the need for a more efficient mode of nutrient acquisition are possible triggers for the transition to necrotrophy. Based on recent findings, we focus on the polyphagous plant pathogen Sclerotinia sclerotiorum. Rather than overwhelming plant foes, S. sclerotiorum has evolved clever means to compromise host recognition and establish disease, resulting in a broad and immensely successful pathogenic lifestyle. The tactics used by this fungus to achieve pathogenic success are being clarified. We propose that the hemi-biotrophic lifestyle may be more temporally and spatially complex than currently depicted, and that combining lifestyle attributes with damage response curves that consider the contribution of both the fungus and the host to pathogenesis, may provide a more holistic manner to view plant pathogens.

Aspartyl protease mediated cleavage of AtBAG6 is necessary for autophagy and fungal resistance in plants

BAG6 processing confers plant immunity via autophagy. 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.

The pathogenic development of Sclerotinia sclerotiorum in soybean requires specific host NADPH oxidases

The plant membrane-localized NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), play crucial roles in various cellular activities, including plant disease responses, and are a major source of reactive oxygen species (ROS). Sclerotinia sclerotiorum is a cosmopolitan fungal pathogen that causes Sclerotinia stem rot (SSR) in soybean. Via a key virulence factor, oxalic acid, it induces programmed cell death (PCD) in the host plant, a process that is reliant on ROS generation. In this study, using protein sequence similarity searches, we identified 17 soybean RBOHs (GmRBOHs) and studied their contribution to SSR disease development, drought tolerance and nodulation. We clustered the soybean RBOH genes into six groups of orthologues based on phylogenetic analysis with their Arabidopsis counterparts. Transcript analysis of all 17 GmRBOHs revealed that, of the six identified groups, group VI (GmRBOH-VI) was specifically and drastically induced following S.xa0sclerotiorum challenge. Virus-induced gene silencing (VIGS) of GmRBOH-VI using Bean pod mottle virus (BPMV) resulted in enhanced resistance to S.xa0sclerotiorum and markedly reduced ROS levels during disease development. Coincidently, GmRBOH-VI-silenced plants were also found to be drought tolerant, but showed a reduced capacity to form nodules. Our results indicate that the pathogenic development of S.xa0sclerotiorum in soybean requires the active participation of specific host RBOHs, to induce ROS and cell death, thus leading to the establishment of disease.

Main and epistatic loci studies in soybean for Sclerotinia sclerotiorum resistance reveal multiple modes of resistance in multi-environments

Genome-wide association (GWAS) and epistatic (GWES) studies along with expression studies in soybean [Glycine max (L.) Merr.] were leveraged to dissect the genetics of Sclerotinia stem rot (SSR) [caused by Sclerotinia sclerotiorum (Lib.) de Bary], a significant fungal disease causing yield and quality losses. A large association panel of 466 diverse plant introduction accessions were phenotyped in multiple field and controlled environments to: (1) discover sources of resistance, (2) identify SNPs associated with resistance, and (3) determine putative candidate genes to elucidate the mode of resistance. We report 58 significant main effect loci and 24 significant epistatic interactions associated with SSR resistance, with candidate genes involved in a wide range of processes including cell wall structure, hormone signaling, and sugar allocation related to plant immunity, revealing the complex nature of SSR resistance. Putative candidate genes [for example, PHYTOALEXIN DEFFICIENT 4 (PAD4), ETHYLENE-INSENSITIVE 3-LIKE 1 (EIL3), and ETHYLENE RESPONSE FACTOR 1 (ERF1)] clustered into salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) pathways suggest the involvement of a complex hormonal network typically activated by both necrotrophic (ET/JA) and biotrophic (SA) pathogens supporting that S. sclerotiorum is a hemibiotrophic plant pathogen.

Development and Evaluation of Glycine max Germplasm Lines with Quantitative Resistance to Sclerotinia sclerotiorum

Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot, is a devastating fungal pathogen of soybean that can cause significant yield losses to growers when environmental conditions are favorable for the disease. The development of resistant varieties has proven difficult. However, poor resistance in commercial cultivars can be improved through additional breeding efforts and understanding the genetic basis of resistance. The objective of this project was to develop soybean germplasm lines that have a high level of Sclerotinia stem rot resistance to be used directly as cultivars or in breeding programs as a source of improved Sclerotinia stem rot resistance. Sclerotinia stem rot-resistant soybean germplasm was developed by crossing two sources of resistance, W04-1002 and AxN-1-55, with lines exhibiting resistance to Heterodera glycines and Cadophora gregata in addition to favorable agronomic traits. Following greenhouse evaluations of 1,076 inbred lines derived from these crosses, 31 lines were evaluated for resistance in field tests during the 2014 field season. Subsequently, 11 Sclerotinia stem rot resistant breeding lines were moved forward for field evaluation in 2015, and seven elite breeding lines were selected and evaluated in the 2016 field season. To better understand resistance mechanisms, a marker analysis was conducted to identify quantitative trait loci linked to resistance. Thirteen markers associated with Sclerotinia stem rot resistance were identified on chromosomes 15, 16, 17, 18, and 19. Our markers confirm previously reported chromosomal regions associated with Sclerotinia stem rot resistance as well as a novel region of chromosome 16. The seven elite germplasm lines were also re-evaluated within a greenhouse setting using a cut petiole technique with multiple S. sclerotiorum isolates to test the durability of physiological resistance of the lines in a controlled environment. This work presents a novel and comprehensive classical breeding method for selecting lines with physiological resistance to Sclerotinia stem rot and a range of agronomic traits. In these studies, we identify four germplasm lines; 91–38, 51–23, SSR51–70, and 52–82B exhibiting a high level of Sclerotinia stem rot resistance combined with desirable agronomic traits, including high protein and oil contents. The germplasm identified in this study will serve as a valuable source of physiological resistance to Sclerotinia stem rot that could be improved through further breeding to generate high-yielding commercial soybean cultivars.

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).

Integrated soybean transcriptomics, metabolomics, and chemical genomics reveal the importance of the phenylpropanoid pathway and antifungal activity in resistance to the broad host range pathogen Sclerotinia sclerotiorum

Sclerotinia sclerotiorum, a predominately necrotrophic fungal pathogen with a broad host range, causes a significant yield limiting disease of soybean called Sclerotinia stem rot (SSR). Resistance mechanisms against SSR are poorly understood, thus hindering the commercial deployment of SSR 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 analyzed 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 (ROS), and importantly, a reprogramming of the phenylpropanoid pathway leading to increased antifungal activities. Indeed, we noted that phenylpropanoid pathway intermediates such as, 4-hydroxybenzoate, 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. Author Summary Resistance to plant fungal pathogens with predominately necrotrophic lifestyles is poorly understood. In this study, we use Sclerotinia sclerotiorum and soybean as a model system to identify key resistance components in this crop plant. We employed a variety of omics approaches in combination with functional studies to identify plant processes associated with resistance to S. sclerotiorum. Our results suggest that resistance to this pathogen is associated in part with an earlier induction of jasmonate signaling, increased ability to scavenge reactive oxygen species, and importantly, a reprogramming of the phenylpropanoid pathway resulting in increased antifungal activities. These findings provide specific plant targets that can exploited to confer resistance to S. sclerotiorum and potentially other pathogens with similar lifestyle.

An inhibitor of apoptosis (SfIAP) interacts with SQUAMOSA promoter-binding protein (SBP) transcription factors that exhibit pro-cell death characteristics

Abstract Despite the importance of proper cell death regulation across broad evolutionary distances, an understanding of the molecular machinery underpinning this fundamental process in plants remains largely elusive. This is despite its critical importance to development, homeostasis, and proper responses to stress. The identification of endogenous plant regulators of cell death has been hindered by the fact that many core regulators of cell death in animals are absent in plant genomes. Remarkably, numerous studies have shown that the ectopic expression of animal prosurvival genes in plants can suppress cell death imposed by many stresses. In this study, we capitalize on the ectopic expression of one of these animal prosurvival genes, an inhibitor of apoptosis from Spodoptera frugiperda (SfIAP), to identify novel cell death regulators in plants. A yeast two‐hybrid assay was conducted using SfIAP as bait to screen a tomato cDNA library. This screen identified several transcription factors of the SQUAMOSA promoter‐binding protein (SBP) family as potential SfIAP binding partners. We confirmed this interaction in vivo for our top two interactors, SlySBP8b and SlySBP12a, using coimmunoprecipitation. Interestingly, overexpression of SlySBP8b and SlySBP12a induced cell death in Nicotiana benthamiana leaves. Overexpression of these two transcription factors also induced the accumulation of reactive oxygen species and enhanced the growth of the necrotrophic pathogen Alternaria alternata. Fluorescence microscopy confirmed the nuclear localization of both SlySBP8b and SlySBP12a, while SlySBP12a was also localized to the ER membrane. These results suggest a prodeath role for SlySBP8b and SlySBP12a and implicate ER membrane tethering as a means of regulating SlySBP12a activity.

An overview of the Sclerotinia sclerotiorum pathosystem in soybean: impact, fungal biology, and current management strategies

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is one of the most important diseases of soybean. Disease management is complicated by the long-term survival of sclerotia in the soil and the absence of resistance in elite, commercial cultivars. Furthermore, the lifecycle of S. sclerotiorum in soybean fields is highly dependent on weather conditions, leading to a highly sporadic occurrence of the disease over seasons and an aggregated distribution within fields. Management relies on a multi-pronged approach of combining partially resistant cultivars with cultural practices, such as altering row spacing and planting population, along with chemical control. These control measures are constrained by economic trade-offs, incomplete efficacy of chemicals, and a lack of understanding of application timing for fungicides. Newer tools have been developed to improve management, such as disease prediction models that can assist farmers in making decisions about fungicide application. This review aims to introduce the Sclerotinia pathosystem in soybean, while covering the complicated biology of S. sclerotiorum that leads to the need for integrated management by soybean farmers.