Research Project: Emerging Stress Challenges and Functional Genomics of Stress Responses in Alfalfa

Location: Molecular Plant Pathology Laboratory

2022 Annual Report

Objectives
1. Identify and develop molecular characterizations of new and emerging disease-causing pathogens in alfalfa to prevent potential threats to alfalfa production. Many known bacterial, fungal, oomycete, nematode, mollicute or viral diseases represent causes of concern for alfalfa industry. In addition, new, emerging and invasive pathogens of uncertain impact pose a serious challenge to the alfalfa improvement. Rapid identification of the causal agents, their characterization at the molecular level and development of sensitive diagnostic assays will reduce yield losses and prompt new insights into practices of alfalfa disease management. 2. Identify genes involved in stress responses in alfalfa to define the genetic basis of resistance and accelerate breeding programs. Emerging disease challenges demand novel approaches to maintain and improve alfalfa production. Understanding molecular mechanisms of stress tolerance is an essential requirement for improvement of alfalfa adaptability and acceleration of breeding programs in increasingly less favorable environmental conditions.

Approach
To fulfill the main goal of Objective 1, the Project will pursue rapid identification of the causal agents, their characterization at the molecular level, and development of sensitive diagnostic assays, aiming to reduce yield losses and to prompt new insights into practices of alfalfa disease management. The approach and research methodology for the detection and/or discovery of new biological and environmental stressors influencing alfalfa quality and productivity will include the following steps critical for the success of the Project: • Specimens collection: alfalfa samples delivery will be negotiated with colleagues, collaborators, alfalfa extension specialists, commercial growers, industry professionals and with diagnostic laboratories-participants of the National Plant Diagnostic Network. Samples will also be collected during on-site visits to alfalfa fields for detection of plant pathogens. • Diagnostics and identification: alfalfa samples will be evaluated by visual assessment, microscopic tools, molecular detection methods (PCR/RT-PCR, LAMP and others), serological assays, and next generation sequencing. • Molecular characterization: identified plant pathogens will be further characterized at the molecular level using comprehensive bioinformatics, molecular and phylogenetic tools. • Development of specific diagnostics tools for pathogen detection, such as pathogen-specific PCRs (conventional, RT-PCR, quantitative PCR, nested and multiplex PCR), molecular hybridization techniques, and serological assays. • Field pathogenomics: integration of genomic data into traditional pathogen surveillance activities. To fulfill the main goal of Objective 2, the Project will use modern experimental and genomic tools combined with computational analysis and systems biology research. Consecutively applied toward each of the plant-pathogen interaction studies, these state-of-the-art methodologies will enable identification and characterization of the genes, involved in stress responses in alfalfa. • Experimental approaches will primarily include the latest high-throughput sequencing methodologies to capture and quantify transcripts present in an RNA extract. • Computational approach will include transcript quantification (estimation of gene and transcript expression); differential gene expression analysis (comparison of expression values among different samples); and functional profiling of RNA-seq data (characterization of the molecular functions or pathways in which differentially expressed genes (DEGs) are involved) • Systems biology research will integrate quantitative metagenomics data into descriptions of genes, pathways, cellular processes and networks to uncover biological insights of alfalfa adaptive responses. • To supplement high-throughput transcriptomics data, the project will attempt to employ global proteomic profiling to identify and characterize proteins involved in alfalfa responses to stress.

Progress Report
Continued study on the discovery and molecular characterization of novel and emerging viruses infecting alfalfa. New symptomatic alfalfa samples from commercial production fields in the U.S. Pacific Northwest have been received and are being investigated to diagnose the causes of diseases. Data collected during the previous several years of research were reviewed, analyzed, and combined into a publication documented in the Accomplishment 1. Progress directly relates to Objective 1. Continued study on the discovery and characterization of novel RNA viruses infecting nematodes and other soil-inhabiting organisms. Metatranscriptomic analysis of soil samples, collected in different regions of the United States resulted in identification of many novel, previously undescribed viral species associated with soil-inhabiting organisms, particularly free-living and plant parasitic nematodes. These are essential steps toward understanding major factors that promote abundance and roles of RNA viral communities in natural environment. Furthermore, nematode-infecting viruses can represent an entirely new resource as a biological control agents. The research has been outlined in the publication documented in the Accomplishment 2. Progress directly relates to Objective 1. Initiated study on using nanopore sequencing for genomic and transcriptomic research related to pathogens’ discovery and whole genome sequencing. Nanopore sequencing is a new generation of sensing technology that offers real-time analysis of DNA or RNA and can sequence ultra-long read lengths. Progress relates to Objectives 1 and 2. Initiated study on alfalfa cyst nematode Heterodera medicaginis, an invasive species recently discovered in U.S. alfalfa. Nematode samples were obtained under APHIS permit and are being propagated to sufficient amounts in order to proceed with genomic sequencing (that is not currently available), research on mechanisms of parasitism, and on possible approaches for controlling the nematode.

Accomplishments
1. Discovery and characterization of new and emerging viruses in alfalfa. Viral infections of alfalfa are widespread in all major cultivation areas and their impact on alfalfa production is often underestimated. The ARS researchers in Beltsville, Maryland, and in Prosser, Washington, discovered, characterized at the molecular level, and developed diagnostic methods for identification of many different viruses infecting alfalfa in the U.S. Pacific Northwest. This research demonstrated that in agricultural settings alfalfa plants are coinfected with numerous viruses representing a ubiquitous and abundant background for all other host-pathogen interactions ranging from synergism to antagonism. The discovery of several novel and emerging viruses raised issues on possible quarantine regulations, coordinated disease-control strategy, disease surveillance, and improved detection technologies to prevent future spread

2. Expanding the RNA virome of nematodes and other soil-inhabiting organisms. Nematodes are among the most abundant animals on Earth. While the assemblage of viruses infecting many arthropods is becoming available, the virome associated with nematodes remains mostly unexplored. In this study, ARS researchers bridged this gap by discovering more than 140 new virus species and demonstrating a remarkable diversity of RNA viruses infecting nematodes and other soil-inhabiting hosts. These findings provided new insights into the manifold diversity of viruses associated with micro-scale soil environment and their important roles in the structural and evolutionary aspects of soil communities. Discovery of new viruses in nematodes can also lead to biotechnological applications for control of plant parasitic nematodes in alfalfa and other crops, and for genetic modification of PPN.

Review Publications
Viera, P., Myers, R.Y., Pellegrin, C., Wram, C., Hesse, C.N., Maier, T.R., Georgios, K.D., Zasada, I.A., Matsumoto Brower, T.K., Danchin, E.G., Baum, T.J., Eves-Van Den Akker, S., Nemchinov, L.G. 2021. Targeted transcriptomics reveals signatures of large-scale independent origins and concerted regulation of effector genes in Radopholus similis. PLoS Pathogens. 17:11. https://doi.org/10.1371/journal.ppat.1010036.
Vieira, P., Subbotin, S.A., Alkharouf, N., Eisenback, J.D., Nemchinov, L.G. 2022. Advancing virus discovery in nematodes and other soil-inhabiting organisms. Virus Evolution. 8(1):1–8. https://doi.org/10.1093/ve/veac019.
Nemchinov, L.G., Irish, B.M., Grinstead, S.C., Shao, J.Y., Vieira, P. 2022. Diversity of the virome associated with alfalfa (Medicago sativa L.) in the U.S. Pacific Northwest. Scientific Reports. 12:8726. https://doi.org/10.1038/s41598-022-12802-4.