Location: Cereal Crops Improvement Research
2024 Annual Report
Objectives
Objective 1. Functionally characterize the pathogen components of the Parastagonospora nodorum-wheat interaction.
Sub-objective 1.A: Functionally characterize the role of SnTox5 in necrosis induction and colonization of the wheat leaf.
Sub-objective 1.B: Characterize the role of SnTox267 in virulence using laser confocal microscopy.
Objective 2. Characterize the infection strategies of Pyrenophora teres, the net blotch pathogen of barley.
Objective 3. Identify, validate, and functionally characterize the Pyrenophora teres genes/proteins important in virulence on barley.
Approach
Fungal diseases pose an economic threat to plant crops throughout the world resulting in billions of dollars in losses annually. A significant amount of work has been done to understand biotrophic host-pathogen interactions. However, less progress has been made to understand the relationship between plants and their necrotrophic pathogens. Here we focus on understanding how the necrotrophic pathogens Pyrenophora teres, causal agent of net blotch of barley, and Parastagonospora nodorum, causal agent of septoria nodorum blotch of wheat, manipulate the host defense to allow pathogen colonization that results in disease. In previous work, we functionally validated several genes/proteins in P. nodorum that contributed to the pathogen’s infection strategy. Here, we will functionally characterize these effectors using modern tools. CRISPR-Cas9-based gene editing will be used to characterize regions of the proteins involved in virulence function, laser confocal microscopy will be used to visualize the role and mode of action of these effector proteins in planta, and comparative transcriptome sequencing of in planta effector infiltrations and inoculations will be used to characterize the host response to these effectors. To characterize the P. teres f. teres-barley interaction, we will identify and validate candidate effector genes conferring virulence on susceptible barley. Candidate genes will be identified and validated using both P. teres biparental and globally collected natural populations that differ in virulence. QTL analysis and genome wide association study (GWAS) analysis will be used to locate candidate genomic regions harboring effector genes. Once effector genes have been validated using CRISPR-based gene disruption, strains with and without the effector genes will be used in inoculation experiments to characterize the mode of action of each effector, ultimately resulting in the understanding of the infection strategy of the pathogen. This work will provide critical knowledge to breeders and other researchers targeting the control of these important diseases.
Progress Report
This report documents progress for Project Number 3060-22000-051-000D, entitled “Host-Pathogen Interactions Affecting Wheat and Barley”.
Septoria nodorum blotch (SNB) (formerly Stagonospora nodorum blotch) on wheat and net form net blotch (NFNB) and spot form net blotch (SFNB) on barley and are three of the most destructive leaf diseases of cereals causing up to 50% yield losses on a susceptible varieties, both in the United States and worldwide unless control measures are taken. In this project we have focused on elucidating the mechanisms of virulence for each pathogen by genetically characterizing the virulence of the pathogen, identifying and validating the genes contributing to virulence, and investigating the mode of action of the corresponding proteins encoded by these genes. We have worked closely with collaborators focused on the host plant's involvement in these interactions.
Progress on Objective 1. Functionally characterize the pathogen components of the Parastagonospora nodorum-wheat interaction.
P. nodorum is a necrotrophic fungal pathogen that induces the host defense response resulting in plant cell death, but instead of stopping the pathogen, the pathogen modulates this host process to provide nutrients for pathogen growth and sporulation. SnTox5 is a necrotrophic effector that is known to induce cell death to the advantage of the pathogen. Pathogen gene expression analysis using SnTox5-producing isolates showed that several classes of genes that are a benefit to pathogen colonization of the plant are upregulated during the colonization period. These pathogen gene classes include chitin binding proteins that protect the fungal (pathogen) cell wall, genes involved in detoxifying or neutralizing host-produced antimicrobial proteins/compounds, proteases used in plant defense manipulation, and cell wall degrading enzymes and necrosis inducing effectors used in nutrient release and acquisition, providing knowledge for how this necrotrophic pathogen is manipulating its host plant.
Gene expression comparisons in the host (wheat) also revealed that the host was manipulated by the Tox5-producing pathogen. Gene classes known to be involved in plant immunity and the negative regulation of cell death were both down regulated, showing that the Tox5-expressing isolates were reducing the immune response to allow the pathogen to colonize but were extending the host-controlled programmed cell death (cellular breakdown) to make nutrients available to the colonizing pathogen.
SnTox267 is another necrosis inducing protein produced by P. nodorum. Laser confocal microscopy and a staining technique that localizes plant-produced reactive oxygen species (ROS) were used to characterize the role of SnTox267 in disease. ROS is an antimicrobial defense response to invading pathogens. We showed that pathogens expressing SnTox267 were more fit to colonize the host plant, and this was at least partly due to the reduction in ROS produced by the plant during early colonization.
Progress on Objective 2. Characterize the infection strategies of Pyrenophora teres, the net blotch pathogen of barley.
Although several studies have characterized the genetics of host resistance/susceptibility, we still have very little understanding of what the pathogen is doing to cause disease and how the host is responding to the pathogen. In previous studies, we mapped and characterized two genes (VR1 and VR2) that conferred virulence on Rika barley. In the current work, we used laser confocal microscopy in conjunction with machine learning to identify the fungal volume more accurately in planta. Expression of VR1 and VR2 independently increased fitness through the buildup of fungal biomass, but also worked synergistically when harbored by the same isolate. Additionally, we used machine learning to accurately identify nuclear breakdown, a hallmark of host-induced cell death, to show that this cell death is delayed by the virulent pathogen until the pathogen has completely colonized the local tissue and is moving to uncolonized areas of the leaf.
Progress on Objective 3. Identify, validate, and functionally characterize the Pyrenophora teres genes/proteins important in virulence on barley.
Pyrenophora teres f. teres and P. teres f. maculata are destructive diseases of barley worldwide. Some understanding of host resistance is available in the literature, however, much less is known about virulence for either of these pathogens.
To cover this gap in understanding in the net form net blotch of barley interaction, a panel of 218 globally collected P. teres f. teres isolates was phenotyped on a 20-line barley differential set containing historical lines used to evaluate P. teres f. teres isolates globally. Full genome sequences were obtained for all 218 isolates for use in mining molecular markers. Together, the genotyping and phenotyping will be used in a genome wide association study to identify genomic regions harboring genes conferring virulence on the various barley lines.
Occasionally, a pathogen of one host will jump to a closely related host, resulting in an emerging disease. Recently, we showed that P. teres f. maculata is not only a pathogen of barley but is also a pathogen of durum wheat, providing a new problem for durum breeders to deal with. Therefore, we evaluated a durum population segregating for resistance/susceptibility to P. teres f. maculata and mapped disease susceptibility to wheat chromosome 2A. To identify the corresponding pathogen virulence, a global collection of 146 P. teres f. maculata isolates were phenotyped on five moderately susceptible to susceptible durum wheat lines. Genome sequencing of the 146 isolates is nearly complete and together, the phenotyping and genotyping of this collection will be used in a genome wide association study to identify the genomic regions and the underlying genes conferring virulence on durum wheat. This work will be useful to durum wheat breeders to better breed resistance to this emerging disease.
Accomplishments
1. Using machine learning to characterize plant-fungal interactions. Laser confocal microscopy has become an essential technique in thoroughly evaluating-plant microbe interactions. However, this work has been somewhat limited to organisms that are amenable to transformation and reliable expression of fluorescent proteins. In previous work, we identified stains that strongly differentiated fungal tissue from plant tissue as well as highlighting the nuclei of healthy cells. ARS researchers in Fargo, North Dakota, used machine learning to generate volume measurements of both fungal hyphae and plant nuclei. Fungal volume was used to track the increase in fungal biomass between timepoints as well as compare fungal biomass between strains of pathogens with and without specific virulence genes. Additionally, since nuclear breakdown is one of the early signs of programmed cell death, cell death could be accurately tracked by nuclear volume to show precisely when cell death was taking place. This machine learning pipeline can be used in most plant-fungal interactions to evaluate pathogen fitness and the timing of plant cell death.
2. A Moroccan population of P. teres f. teres overcomes a commonly used source of barley resistance. Net form net blotch (NFNB) is one of the most destructive fungal pathogens of barley globally, with typical losses ranging from 10-40% without the use of fungicides or resistant cultivars. Some barley lines harbor a highly effective resistance to the causal pathogen P. teres f. teres and this resistance source referred to as Rpt5 is commonly used in breeding programs to guard against NFNB. ARS researchers in Fargo, North Dakota, assembled and evaluated a global population of P. teres f. teres on a barley mapping population segregating for the Rpt5 resistance gene. A subset of a Moroccan collection were the only isolates to overcome the Rpt5 resistance, indicating that Rpt5 is vulnerable globally. This information is critical to breeders using the Rpt5 resistance in cultivar development.
Review Publications
Taliadoros, D., Feurtey, A., Wyatt, N.A., Gladieux, P., Friesen, T.L., Stukenbrock, E. 2024. Population genomic analyses and demography inference show recent emergence and dispersal of barley pathogen coinciding with crop domestication and cultivation history. PLoS Genetics. https://doi.org/10.1371/journal.pgen.1010884.
Kariyawasam, G., Nelson, A., Williams, S., Solomon, P., Faris, J.D., Friesen, T.L. 2023. The necrotrophic pathogen Parastagonospora nodorum is a master manipulator of wheat defense. Molecular Plant-Microbe Interactions. https://doi.org/10.1094/MPMI-05-23-0067-IRW.
Fiedler, J.D., Friesen, T.L., Rouse, M.N., Jin, Y., Green, A.J., Mergoum, M., Frohberg, R., Underdahl, J., Walz, A., Seiland, T. 2023. Registration of “ND Frohberg” hard red spring wheat. Journal of Plant Registrations. 17:385-396. https://doi.org/10.1002/plr2.20291.
Li, J., Wyatt, N.A., Skiba, R., Kariyawasan, G., Richards, J., Effertz, K., Rehman, S., Brueggemann, R., Friesen, T.L. 2024. Variability in chromosome 1 of select Moroccan P. teres f. teres isolates enables isolates to overcome a highly effective barley chromosome 6H source of resistance. Molecular Plant-Microbe Interactions. https://doi.org/10.1094/MPMI-10-23-0159-R.