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

Location: Molecular Plant Pathology Laboratory

2019 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
A separate annual report (8042-21000-271-00D) has been submitted on the overlapping bridge period between termination of the old project on 04/08/2018 and start of the new project on 03/24/2019). Initiated study on global proteomic profiling and identification of stress-responsive proteins in alfalfa against agriculturally important migratory root lesion nematode Pratylenchus penetrans. The research will enable identification of host proteins involved in defense response. Progress directly relates to Objective 2. Continued studies on the discovery and functional characterization of the virulence effectors of the root lesion nematode P. penetrans. Knowledge of the nematode parasitism genes can be applied toward alfalfa breeding programs or used in the development of new nematicides. Progress directly relates to Objective 2. Continued studies on the discovery of new viral pathogens infecting alfalfa. Systematic surveys of publicly available alfalfa transcriptomic data are being performed to identify and characterize novel or not previously identified in alfalfa emerging plant viruses. Progress directly relates to Objective 1. Alfalfa samples with diseased symptomatology are being received from our collaborators under Animal and Plant Health Inspection Service (APHIS) permits. The samples are investigated in the laboratory to diagnose the causes of diseases. Progress directly relates to Objective 1. Continued working of the development and improvement of the virus-based vector for alfalfa functional genomic studies. Progress directly relates to Objective 2.

Accomplishments
1. Discovered a novel species of RNA virus identified in the migratory root lesion nematode Pratylenchus penetrans. The root lesion nematode Pratylenchus penetrans is a migratory species affecting agriculturally important crops worldwide, including alfalfa. The root lesion nematode virus (RLNV) is the first virus identified in the migratory nematode species of the genus Pratylenchus. The effect of the virus infection on the viability and development of the nematodes has yet to be clarified. The research is significant because it provides insights into the nematode biology and may result in application of virus infection for pest control strategies.

2. For the first time, identified a strain of Cycas necrotic stunt virus (CNSV) in publicly available transcriptomic datasets of alfalfa. Prior to this work, the virus was not reported to infect alfalfa. Cycas necrotic stunt virus was first detected in the gymnosperm Cycas revoluta in Japan where it caused severe leaves abnormalities. The virus identified in alfalfa by global surveys of transcriptomic datasets represents a new strain of CNSV adapted to alfalfa, for which the name CNSV-A was proposed. The research is significant because the biology of this emerging virus in alfalfa is not known and requests further investigation.

3. For the first time, identified a strain of cnidium vein yellowing virus in publicly available transcriptomic datasets of alfalfa. Prior to this work, the virus was not reported in alfalfa and was only known to infect cnidium, a medicinal plant species (Cnidium officinale) in Korea, where infected cnidium plants displayed yellowing leaf symptoms. The research is significant because the biology of this emerging virus in alfalfa is not known and requests further investigation.

Review Publications
Nemchinov, L.G., Francois, S., Roumagnac, P., Hammond, R., Mollov, D.S., Filloux, D. 2018. Characterization of alfalfa virus F, a new member of the genus Marafivirus. PLoS One. 13(9):e0203477. https://doi.org/journals.plos.org/plosone/article?id=10.1371/journal.pone.0203477.
Nemchinov, L.G., Lee, M., Shao, J.Y. 2018. First report of alphapartitiviruses infecting alfalfa (Medicago sativa) in the United States. Genome Announcements. https://doi.org/mra.asm.org/content/7/21/e01266-18.
Vicente, C.S., Nemchinov, L.G., Mota, M., Eisenback, J.D., Kamo, K.K., Vieira, P. 2019. Identification and characterization of the first pectin methylesterase gene discovered in the root lesion nematode Pratylenchus penetrans. PLoS One. 14 (2):e0212540. https://doi.org/10.1371/journal.pone.0212540.
Vieira, P., Nemchinov, L.G. 2019. A novel species of RNA virus associated with root lesion nematode Pratylenchus penetrans. Journal of General Virology. https://doi.org/10.1099/jgv.0.001246#tab2.