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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Molecular Plant Pathology Laboratory » Research » Research Project #424531

Research Project: Regulation of Gene Expression in Alfalfa Development and Stress Tolerance

Location: Molecular Plant Pathology Laboratory

2018 Annual Report


Objectives
1. Improve the efficiency of developing alfalfa with greater tolerance to biotic and abiotic stresses by characterizing gene-stress responses and pathways. Biotic and abiotic stresses cause significant yield losses in alfalfa and greatly reduce the crop’s productivity. Understanding the molecular mechanisms of stress tolerance and the ways by which stress-responsive genes are regulated is essential for improvement of alfalfa adaptability and breeding programs. 2. Aid plant breeders in improving alfalfa productivity and adaptability by implementing genetic and genomic approaches to improve traits related to biotic and abiotic stress tolerance, including, but not limited to, root-knot nematodes and salinity tolerance. Data on stress-responsive genes obtained in this study and other information on alfalfa genomics will be used to identify molecular markers associated with resistance and adaptation to abiotic and biotic stresses in alfalfa.


Approach
The research project will identify stress-responsive gene-candidates in alfalfa and assign them to cognate functional groups related to specific stress responses. It will quantify and confirm roles of the selected genes in adaptation to abiotic and biotic stresses and in regulation of stress responses. Sequence polymorphism in genes underlying stress tolerance will be delineated and molecular markers associated with resistance and adaptation of alfalfa to biotic and abiotic stresses developed. Markers will be validated through cooperative research collaborations.


Progress Report
This is the final report for the project 8042-21000-268-00D. The replacement project, entitled “Emerging Stress Challenges and Functional Genomics of Stress Responses in Alfalfa” is going through the scheduled OSQR Peer Review. Most of the planned experiments were completed prior to the project’s expiration date and new experiments corresponding to the replacement project were initiated. During the life of this project, extensive knowledge on expression and regulation of genes associated with alfalfa development and stress tolerance was generated. Multiple projects on global transcriptomic studies of alfalfa responses to stress were undertaken and completed. Gene-candidates involved in alfalfa salinity tolerance, alfalfa resistance to root-knot nematode and to a causal agent of bacterial stem blight were proposed and polymorphic molecular markers identified. Alfalfa transcription factors, proteins that govern organismal development and responses to stress were identified, characterized and integrated into publicly accessible database. The overall impact of these accomplishments is that they opened new opportunities for alfalfa genetic improvement and new molecular targets involved in regulation of stress responses in the crop. As a byproduct of the mainstream research theme, a new research direction related to diagnostics and characterization of novel diseases in alfalfa has branched off the old project. New biological and environmental stressors influencing alfalfa quality and productivity constantly emerge in the changing environment. Besides, dynamic environment elicits variations in the behavior and distribution of known pathogens. Development of stress-tolerant alfalfa cultivars requires an in-depth knowledge of these risk factors and the genetic basis of host responses to them. The replacement project combines a critical objective of the expired program plan, aimed at understanding the molecular mechanisms of stress tolerance in alfalfa, with a new objective dedicated to the discovery, molecular characterization and diagnostics of new and emerging disease-causing pathogens. Fulfillment of these objectives will help to prevent potential threats to alfalfa production and to further delineate the genetic basis of resistance needed for acceleration of breeding programs.


Accomplishments
1. Identified genes involved in salinity tolerance in alfalfa. Salinity is one of the major physical stresses affecting alfalfa productivity. A comprehensive analysis of alfalfa’s response to salt stress was performed. Scientists at ARS in Beltsville, Maryland, proposed gene-candidates involved in alfalfa salinity tolerance and identified molecular markers to detect those genes so they could be studied. Prior to this work, there were no published studies about changes in gene expression patterns of alfalfa in response to salinity stress. The results of this study directly benefited scientists involved in alfalfa research, providing them with new genomic insights into mechanisms of stress tolerance, an up-to-date list of genes involved in response to salinity, and new information for molecular breeding programs.

2. Identified natural antisense transcripts in alfalfa associated with response to salt stress. Natural antisense transcripts (NAT) are a group of RNAs found in cells and recognized as important regulators of gene expression during a cell’s responses to the environment. No information about NAT in alfalfa was available prior to this research. ARS scientists in Beltsville, Maryland, demonstrated that NATs are involved in regulating salt tolerance in alfalfa. The research provided new knowledge about expression and regulation of genes associated with alfalfa development and stress tolerance.

3. Identified and characterized alfalfa transcription factors and developed an open-access database. Transcription factors (TFs) are proteins that govern an organism’s development and response to the environment and only scattered information about separate alfalfa TFs was available prior to this work. A computer-based analysis of alfalfa RNA data and by ARS scientists in Beltsville, Maryland, allowed for identification of the TFs, which were characterized into groups and analysis of the evolutionary development and diversification of the largest TF families showed that their composition is distinct from TFs found in other higher plants, suggesting they may have unique biological roles in alfalfa. The study presents an essential resource addressing key proteins in the regulation network of genes in alfalfa. For the first time, alfalfa TFs were systematized and integrated into a simple, publicly accessible database named AlfalfaTFDB. Transcription factor datasets can be retrieved using a convenient search tool. The database provides users with an overview of all annotated alfalfa TF families, a reference to the description of each family, identified RNA and its derived protein sequences.

4. Identified candidate genes involved in alfalfa resistance to root-knot nematodes. The root-knot roundworm (nematodes) Meloidogyne species (RKN) are widely distributed and economically important parasites impacting crops and potentially inflicting significant damage to alfalfa. Prior to this work, no published studies were available about changes in the gene expression pattern in alfalfa infected with RKN or any other parasitic nematode of plants. A comprehensive analysis of the changes in gene expression patterns during alfalfa infection by southern root-knot nematode by ARS scientists in Beltsville, Maryland, led to the identification of genes involved in resistance to RKN. Mechanisms of alfalfa resistance against RKN in alfalfa were proposed that can be a resource for improvement of alfalfa adaptability to enhance breeding programs. The research led to a collaboration with Virginia Polytechnic Institute and State University (Virginia Tech) on another major pest in alfalfa, the root lesion nematode Pratylenchus penetrans, for which no commercially certified varieties with resistance are currently available.

5. Identified putative genes and mechanisms for alfalfa resistance to bacterial stem blight disease. The bacterium Pseudomonas (P.) syringae infects many agricultural crops, including alfalfa. Little is known about alfalfa interactions with this disease-causing bacterium. In this research, for the first time, ARS scientists in Beltsville Maryland, found distinct gene expression patterns in alfalfa plants that exhibited resistance or susceptibility to the bacterium, and identified alfalfa genes whose expression pattern changed during in the course of infection. Key genes and biological processes involved in host resistance were proposed. Research on alfalfa-P. syringae interactions made it possible to propose mechanisms of alfalfa resistance to bacterial stem blight disease and suggested a source of germplasm for developing resistant cultivars. This achievement will enable breeders to identify potential casual loci in alfalfa breeding populations.

6. Identified genes involved in the survival of bacteria in plants. One of the possible ways of bacterial survival in different hosts it to enter a low metabolic state, meaning the bacteria can survive but not multiply, also referred to as viable but nonculturable (VBNC) in human medicine. The biological mechanisms underlying VBNC are largely unknown. ARS scientists in Beltsville, Maryland, conducted research to define detained gene expression patterns of Pseudomonas syringae bacteria in the VBNC state. The goal was to shed light on the biological relevance of microbial dormancy in overcoming unfavorable environmental conditions and has application to a wide range of crop plants infected by P. syringae, including alfalfa.

7. Development of a virus vector for functional genomics studies in alfalfa. Viruses have the ability to infect cells and release their genetic material that can halt the production of protein from RNA. This is referred to as viral-induced gene silencing (VIGS). Genetic material impacting gene expression can be introduced into a virus, which is then used as a vector to dissect the function of genes; this is one of the most widely used molecular tools in plants. It has not been applied in alfalfa research due to lack of an appropriate viral vector. Availability of a specific VIGS vector would greatly facilitate studies of the key genes involved in various aspects of alfalfa development and adaptation to the environment. In this project, ARS scientists in Beltsville, Maryland, determined the first complete nucleotide sequence and genome structure of a long-known but poorly characterized alfalfa virus. The virus was modified to generate a viral vector for gene silencing and expression for foreign proteins in alfalfa. For the first time, a breakthrough VIGS technology has been attainable for alfalfa genomic studies. This novel methodology will help to gain critical insights for alfalfa breeding programs.

8. Functionally characterized plant gene critical for overall development and responses to the environment. The genome contains genes from which RNA is produced and ultimately proteins that are important for biological functions in an organism. This is a fundamental mechanism in all living cells. In plants, the production of RNA from all genes requires five factors (I, II, III, IV, V) called nuclear RNA polymerases that have distinct functional roles. RNA polymerase III produces the RNA from genes essential for basic cell function. In this work, using modern molecular tools, ARS scientists from Beltsville, Maryland, demonstrated for the first time that a protein, RPC5, is critical for the proper function of RNA polymerase III, for normal plant development and for responses to the environment. Fundamental knowledge of the molecular biology of the cell was obtained. The acquired knowledge will improve understanding of the basic molecular processes in eukaryotic cells, aiding scientists researching cell biology.

9. Discovered, characterized and developed diagnostics methods for new and emerging viruses in alfalfa. Viral infections of alfalfa are widespread in major cultivation areas and their impact on alfalfa production may be underestimated. In recent years, new and emerging viral alfalfa diseases with the capacity to cause serious yield losses were discovered. In this work, ARS scientists from Beltsville, Maryland, in a team effort with ARS’ National Germplasm Research Laboratory and with France’s National Institute for Agriculture (INRA), discovered and characterized two new plant viral species and a new viral strain. Diagnostics methods for sensitive detection of the pathogens were developed based on the sequence information. The discovery of these viruses raises critical issues in quarantine and alfalfa resistance to the pathogens. Diagnostic tools developed in this work may be used to screen U.S. commercial varieties and prevent introduction of the newly discovered pathogens to the U.S.

10. First evidence that a previously unseen virus might infect alfalfa varieties in the U.S. The partitiviruses are associated with persistent infections of fungal, protozoan and plant hosts. Their relationship with plant hosts is not well understood. While examining gene expression data derived from two U.S. alfalfa check cultivars, we found that 95% of alfalfa plants were infected with partitivirus recently reported in Korea. The complete RNA nucleotide sequence of the U.S. isolate of the virus was obtained. Since partitiviruses are transmitted via seeds, the seeds of these standard check U.S. cultivars, commonly used in research and breeding, were most probably infected with the virus. Biological significance or any negative effects of the virus on alfalfa are currently unknown and require further investigation. This is a first identification of the virus in alfalfa samples in the U.S. (manuscript in preparation).

11. Developed advanced understanding of alfalfa interaction with root lesion nematode Pratylenchus penetrans. Pratylenchus penetrans is a migratory roundworm (nematode) species that attacks a broad range of the crops, including alfalfa. Currently, there are no commercially certified varieties with root lesion nematode resistance. A comprehensive study of the gene expression pattern and analysis of changes in the pattern during alfalfa-nematode interaction was performed from which candidate genes involved in resistance to the pathogen were identified. The acquired knowledge will improve our understanding of processes involved in nematode pathogenicity and host resistance, benefitting scientists involved in breeding alfalfa for improved resistance to diseases.


Review Publications
Nemchinov, L.G., Shao, J.Y., Lee, M., Postnikova, O.A., Samac, D.A. 2017. Resistant and susceptible responses in alfalfa (Medicago sativa) to bacterial stem blight caused by Pseudomonas syringae pv. syringae. PLoS One. 12(12):e0189781.