Source: SOUTH DAKOTA STATE UNIVERSITY submitted to
UNDERSTANDING AND IMPROVING BIOTIC AND ABIOTIC STRESS RESISTANCE IN CROP PLANTS THROUGH PROTEOMICS AND BIOTECHNOLOGICAL APPROACHES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
0220455
Grant No.
(N/A)
Project No.
SD00H345-09
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Sep 1, 2009
Project End Date
Sep 30, 2014
Grant Year
(N/A)
Project Director
Rohila, J.
Recipient Organization
SOUTH DAKOTA STATE UNIVERSITY
PO BOX 2275A
BROOKINGS,SD 57007
Performing Department
Biology & Microbiology
Non Technical Summary
Biotic (e.g. pathogens) and abiotic (e.g. drought) stresses are a major limiting factors for plant growth, development and crop productivity in South Dakota and elsewhere. However, every plant has some degree of innate tolerance or resistance to such stresses but for a successful crop we need to improve the tolerance levels of these crop plants to keep pace with the future food demands. Our hypothesis is that the stress-tolerant phenotype is a result of differential expression of unique proteins in resistant cultivars as compare to the susceptible ones to protect them during stress periods in plant's life. To develop better crop plants for our future needs, discovery of these unique proteins and understanding of the stress-tolerance mechanisms at molecular level is very important. Insights of the stress tolerance proteins- "the key players inside the cell"; will assist our plant breeders in developing better cultivars for our state needs. We can modify these key molecular players to enhance crop's stress tolerance. Discovery of such proteins that are involved in regulating biotic and abiotic stresses will help plant breeders to improve the crop plants using both conventional breeding and transgenic technologies. At present there is much progress in transformation of plants like arabidopsis, tobacco and rice but crops like wheat and soybean have lagging behind. Wheat and soybeans are still considered recalcitrant plants in terms of their genetic transformation potential. Hence, establishing efficient genetic transformation protocols are very important. As a result my research program involve studying both biotic and abiotic stresses to elucidate the mechanism of crop stress tolerance and improving stress tolerance through application of biotechnological means like genetic transformation. The molecular approach will help in understanding the stress tolerance mechanism and the biotechnology approach will be useful in crop-breeding programs.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2031549103010%
2031549104020%
2031820103010%
2031820104010%
2061549103020%
2061549104010%
2061820103010%
2061820104010%
Goals / Objectives
The broad and long term goal of this research project is to understand the complex mechanism of signal transduction, differential protein expression and protein-protein interactions during abiotic and biotic stress occurrences in a plant's life, with an ultimate goal of improving the South Dakota crops specifically for the stresses in the investigation. Specific research objectives are: 1. Understanding of major proteins and their interactions involved in abiotic (e.g. drought) and biotic (e.g. pathogen) stress tolerance mechanisms in major crop plants. 2. Genetic engineering of major crop plants for enhanced biotic and abiotic stress tolerance. It is expected that the proposed research will result in great understanding of biotic and abiotic stress tolerance mechanism in crop plants of interest. The proposed biotechnology component of the project has a potential to develop stress tolerant lines for use in plant breeding programs.
Project Methods
Approach: Proteome is the translated version of a genome and our hypothesis is that numerous proteins of a proteome are expressed differentially in different cultivars/lines which make them resistant or susceptible for a particular stress environment. In this direction the first step is to understand the differential expression of these proteins in different lines resulting from various biotic/abiotic stresses. In this regard we plan to use cutting-edge quantitative proteomic techniques like iTRAQ or 2D-DIGE along with Mass Spectrometry to get insights of differential plant proteome under various stresses. Next, we aim to understand potential in-vivo interacting partners for these proteins by TAP-tag technology. It is a tandem affinity approach coupled with mass spectrometry for the identification of interacting protein partners inside the plant cell (ie. in-planta). In this direction our overarching goal will be to map the interactome for such useful proteins and use those "signature proteins" in the future crop improvement programs as well as in the transgenic approach. Further, our proteomic approach explained above will provide potential candidate proteins and we plan to verify them by a transgenic approach through in-planta expression. The transgenic plants produced in the lab will be tested for transgene presence, expression and functionality via southern blot, northern blot, real-time PCR, western blots and green house studies. For studying stress tolerance, the transgenic plants will be screened in green houses and the field by plant breeders, for physiological and agronomical characters and for protein-protein interactions.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: The target audiences of this project were US wheat, soybean and rice farmers. The undergraduate and graduate students were also among the target audience. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project provided excellent opportunities for professional development to three graduate students and two undergraduate students while working in the project leader's lab. In 2013, as a result of this project we were able to attract 3 international visiting scientists from (Algeria, South Korea, and India)to our lab. How have the results been disseminated to communities of interest? The results were disseminated via formal class teaching , seminars, poster presentations, peer-reviewed journal articles, writing book chapters, and by inviting visiting scientists and giving the required training to them. What do you plan to do during the next reporting period to accomplish the goals? I plan to wrap up this project so I can start a new project that will bebuildup on the success of this project.

Impacts
What was accomplished under these goals? Last year we already accomplished our objective 2. This year we have made a significant progress on the objective 1. In this direction we have identified proteins involved in lignin biosynthesis in corn (the results are already published in the "Journal of Proteomics"). In wheat plant we have identified 90 proteins that are differentially expressed as a result of drought and heat stress (data is under reevaluation as the graduate student is working on his Ph.D. thesis). Moreover an interesting discovery was made for Fusarium attack on wheat plant. Here a set of six genes (proteins) were found to be responsible for providing scab resistance to wheat. Out of six two genes were revalidated by an independent technique (quantitative RT-PCR) and by an independent lab (Yang Yen). This discovery was presented at national scab forum in 2013. We are working on a publication for this data.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Comparative Proteomics Analysis by DIGE and iTRAQ Provides Insight into the Regulation of Phenylpropanoids in Maize. 2013. M. L. Robbins, A. Roy, P. H. Wang, I. Gaffoor, R. S. Sekhon, M. Buanafina, J. S. Rohila, S. Chopra. Journal of Proteomics 93, 254-275.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Identification, nomenclature and evolutionary relationships of Mitogen Activated Protein Kinase (MAPK) gene family in soybean. 2013. A. Neupane, M. P. Nepal, S. Piya, S. Subramanian, J.S. Rohila, R. N. Reese. Evolutionary Bioinformatics 9, 363.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Proteomics: a biotechnology tool for crop improvement. Eldakak, M., S.I. Milad, A.I. Nawar, and J.S. Rohila. 2013.Frontiers in Plant Science.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: INPPO Actions and Recognition as a Driving Force for Progress in Plant Proteomics: Change of Guard, INPPO Update, and Upcoming Activities. GK Agrawal, D Job, T Kieselbach, BJ Barkla, S Chen, R Deswal, S L�thje, RS Amalraj, G Tanou, BK Ndimba, R Cramer, W Weckwerth, S Wienkoop, M J Dunn, ST Kim, Y Fukao, M Yonekura, L Zolla, JS Rohila, R Waditee?Sirisattha, A Masi, T Wang, A Sarkar, R Agrawal, J Renaut, R Rakwal. Proteomics 13 (21): 3093-3100.
  • Type: Book Chapters Status: Accepted Year Published: 2014 Citation: Genomic Methods for Improving Abiotic Stress Tolerance in Crops. Kim, D.W.; Agrawal, G.K.; Rakwal, R.; Ahmed, S.; Rohila J.S. 2013. Plant Biotechnology - Experience and Future Prospects. Editors: Dr Agnes Ricroch, Dr Surinder Chopra, Dr Shelby Fleischer. Publisher: Springer. Accepted (In Press).
  • Type: Book Chapters Status: Accepted Year Published: 2014 Citation: Drought Resistance in Small Grain Cereal Crops. Moustafa Eldakak, Mukhtar Ahmed, Muhammad Asif, Sanaa I. M. Milad, Ali I. Nawar, Zohra Aslam Ali, Aakash Goyal, Jai S. Rohila. 2014. Handbook of Plant and Crop Physiology, Third Edition, Editor: Mohammad Pessarakli) CRC Press. Accepted (In Press).
  • Type: Book Chapters Status: Accepted Year Published: 2014 Citation: Soil and Crop Management Practices for Sustainable Agriculture. Sandeep Kumar, Rajesh Chintala, Jai S. Rohila, Eric G. Mbonimpa, Tom Schumacher and Aakash Goyal. 2014. Sustainable Agriculture Reviews. Accepted (In Press).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Discovery and revalidation of scab responsive genes in wheat by 2D-DIGE and Q-PCR. M Eldakak, A Roy, Y Zhuang, K Glover, S Ali, Y Yen, JS Rohila. 2013 at National Fusarium Head Blight Forum.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: 1. In Vitro manipulations of wheat for genotype-independent genetic engineering via Agrobacterium tumefaciens. M.M. Shirke, S. Rohila, K. Buehner, M. Eldakak, J.S. Rohila. 2013 Poster presentation at The Society for In Vitro Biology Annual Meeting, Rhode Island.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Determination of Fusarium graminearum chemotypes prevalent on oat, rye heads, and wheat roots in South Dakota. S Ali, S Abdullah, P Gautam, E Byamukama, JS Rohila, M Eldakak, K Glover, J Gonzalez. 2013 Proceedings of National Fusarium Head Blight Forum.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Evaluation of global spring wheat germplasm for resistance to tan spot Pyrenophora tritici-repentis race 1.S Ali, S Abdullah, K Glover, JS Rohila. Phytopathology 103 (6): 4-5.


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: During this year we have completed one of the two objectives of the project, which is genetic engineering of wheat. This success of this objective comes with a lot of hard work from the graduate student and undergraduate students especially Manali Shirke and Kim Buehner who put many untiring hours to the project. The wheat is a recalcitrant plant species for genetic manipulations by Agrobacterium tumefaciens for some unknown reasons. This is the reason most of the wheat transformations are done by gene gun. Three years ago we took this challenge and by performing calculative step-wise improvements in the protocol achieved this objective successfully. In this process we have developed the right strain of Agrobacterium, right type of media for transformation including the type of sugars, hormones, and concentrations of various salts. During the year of 2012, one graduate student (Manali Shirke), and three undergraduate students (Kim Buehner, Nina Kamrowski, and Sara Glisczinski) were trained for with hands-on for this technology. One undergraduate (Kim Buehner) student was able to achieve high distinction in her Honors College education because of this successful project. By achieving transformation capabilities in cereals, we have established collaboration with Dr. Justin Farris (USDA, North Dakota) and helped him with the production of transgenic rice plants with Tsn1 gene. We provided 6 transgenic lines for Tsn1 gene to him. Towards the second objective of the project, a graduate student (Moustafa Eldakak) and a visiting scientist (Dr. Ansuman Roy) worked on the proteomics aspect of this hatch project during this year. As a result we have identified 96 different proteins, whose expression levels have been affected by drought, heat, drough+heat and/or fusarium infection. These are interesting proteins for the downstream experiment to study further in greater details. This work has yielded us collaboration with Penn State University (Dr. Surinder Chopra). In this collaborative project we are studying molecular factors, especially proteins that are responsible for pericarp color change and bented stem phenotype by an epigenetic mutation in a wild type allele. Dr. Roy is in the process of writing his peer-reviewed research paper and for its submission to a peer-reviewed journal very soon. The graduate student is advancing the work on refining the transcriptomics, proteomic and metabolomics technologies, and to discover more genes that are responsive to biotic and abiotic stress environments. The overarching goal of this project is to use these genes in the improvement of South Dakota crop plant's for variable climate change. During the year 2012, we have completed one objective of the project and are continuing on the second objective with a great pace. Other than Manali and Moustafa, two graduate students (Prateek Tripathi and Achal Neupane) were also trained for transcription factor and MAPK studies. The results of the project are continually presented by student and faculty at state level scientific meeting and national/international level meetings. PARTICIPANTS: People involved on this project during this period include Ansuman Roy (visiting scientist), Manali Shirke (graduate student), Moustafa Eldakak (graduate student), Achal Neupane (graduate student), Prateek Tripathi (graduate student), Kim Buehner (undergraduate student), Nina Kamrowski (undergraduate student) and Sara Glisczinski (undergraduate student). TARGET AUDIENCES: The target audiences of this project are US wheat, soybean and rice farmers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Transforming wheat by Agrobacterium was one of the great challenges because of its recalcitrant nature. During this year we have tested, 20 different genotypes of wheat along with four different Agrobacterium strains for their virulence to the wheat calli and by modifying the medium, growth, and infection conditions we were successful to hit the right Agrobacterium strain with right growth conditions and with the right Agrobacterium infection media. This knowledge has greatly enhanced our understanding for the wheat calli and Agrobacterium infection chemistry. The modification in all these parameters yielded to a successful Agrobacterium-mediated wheat transformation events. The next step was more interesting when we moved the regenerated transgenic plantlets to the soil, and for their histochemical and molecular analysis. Not all regenerated plants showed positive event for the genetic transformation, it do come with false positives. That means some regenerated plants were able to pass the selection pressure but were not transgenic plantlets. All the positive plants showed the presence of transformed gene during the histochemical assay. Since the plants were transformed with GUS gene, an enzyme whose substrate is X-Gluc. For this objective, we have used a constitutive promoter to drive this gene in plant system; as a result the gene should be expressed in all the plant tissues. For histochemical assays we used the leaves of regenerated transgenic plants for GUS staining. A number of transgenic plants show blue staining confirming the successful integration of T-DNA into wheat cells and the regeneration of a transformed plant from that particular cell, which expressed a positive GUS expression in regenerated plant's leaves. These plants were later subjected to re-confirmation by PCR technology. Since wild type plants lack that gene but the transgenic plants must have it, it was selectively amplified only in the PCR tube which contained the DNA from the positive transgenic plants. This independent experiment re-confirmed the success of the Agrobacterium mediated transformation of the wheat cells. These results have a great impact on wheat biotechnology industry. The data analysis of the experiments for second objective are in process but the preliminary data coming out is very exciting. By utilizing proteomics and mass spectrometry technology we have identified 7 Fusarium responsive genes. This data eventually will be very useful in tackling the wheat scab disease in the state. We are in the process of revalidating these genes. Towards the broader goal of generating new knowledge and enhancing the understanding of the drought tolerance, disease resistance mechanism in wheat and soybean, we are utilizing systems biology approach by inclusion of transcriptomics, proteomics and metabolomics platforms. We have generated a vast amount of data from these experiments, which is under analyses at this moment.

Publications

  • Proteomic dissection of near isogenic lines for the discovery of scab responsive genes of wheat. Eldakak, M., A. Roy, Y. Zhuang, K. Glover, S. Ali, Y. Yen, and J.S. Rohila. 2012 Poster presentation at National Fusarium Head Blight Forum, Orlando, FL. pp 133.
  • Shirke, M., S. Rohila, N. Kamrowski, S. Glisczinski, K. Buehner, A. Roy, M. Eldakak, and J. S. Rohila. 2012. Exploiting the potential of genetic engineering to improve wheat for South Dakota. Poster presentation at 7th Annual SD Biotechnology Summit, Sioux Falls, SD.
  • Neupane, A., S. Piya, R. N. Reese, J.S. Rohila, S. Subramanian, and M. P. Nepal. Identification of Mitogen Activated Protein Kinase Family Members in Soybean. Botany 2012, Poster presentation at Botany 2012 - The Next Generation Conference July 7- 11. Columbus, OH, USA.
  • Neupane, A., S. Piya, S. Subramanian, J. S. Rohila, R. N. Reese, and M. P. Nepal. A Nomenclatural Conundrum: Applying Existing Nomenclature to the Identification of Soybean (Glycine max) MAP Kinase Genes. Poster presentation at ASPB Midwestern Section Meeting 2012, March 24-25. Lincoln, NE, USA.
  • Gibberrella zeae Chemotype Diversity on Moderately FHB Resistant Wheat Genotypes in South Dakota. Ali, S., M. Eldakak, P. Gautam, K. Glover, J.S. Rohila, J. Gonzalez, W. Berzonsky. In: Canty, S., A. Clark, A. Anderson-Scully and D. van Sanford (Eds.), Proceedings of the 2012 National Fusarium Head Blight Forum. Pages 115-117.
  • The WRKY transcription factor family in Brachypodium distachyon. 2012. Tripathi, P. R.C. Rabara, T.J. Langum, A.K. Boken, D.L. Rushton, D.D. Boomsma, C. I. Rinerson, J. Rabara, R. N. Reese, X. Chen, J. S. Rohila, P. J. Rushton. BMC Genomics 13 (1), 270.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: During the second year of the project two Ph.D. students, one visiting scientist and one undergraduate student were trained. The project was mainly focused on studying the drought stress responsive proteins at molecular levels and improving wheat transformation efficiencies. The results of the project were presented at one state level scientific meeting and at one international level meeting. PARTICIPANTS: People involved on this project in my lab during this period include Ansuman Roy (visiting scientist), Manali Shirke (graduate student), Moustafa Eldakak (graduate student) and Kim Buehner (undergraduate student). Dr. Chopra and his lab members at Pennsylvania State University, University Park, PA were collaborators during this period. TARGET AUDIENCES: The target audiences of this project are US wheat and corn researchers. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
During the second year of the project we have done preliminary proteomics experiment in corn and wheat. The focus of these experiments was to understand the drought responsive phenomenon and to discover biomarkers for drought tolerance in plants. We have identified a few differentially expressed proteins in wheat as a result of drought occurrence. In corn, our proteomic studies were in collaboration with Dr. Surinder Chopra (Pennsylvania State University) using Ufo1-1 mutant corn lines. This study leads to the identification of abiotic stress-associated proteins in corn. Revalidation of the proteins identified in corn is underway.

Publications

  • Roy, A., Yang, Z., Robbins, M., Chopra, S. and Rohila, J.S. 2011. Proteome analysis of maize pericarp by DIGE and ITRAQ. In: 96th Annual Meeting of South Dakota Academy of Science at Oacoma, SD, USA.
  • Yang, Z., Robbins, M., Chopra, S., Roy, A., Rohila, J.S. 2011. Pericarp proteome reveals that Unstable factor for orange1 plants exhibit altered expression of stress-associated genes. In: 53rd Annual Maize Genetics Conference at St. Charles, Illinois.
  • Roy, A., Rushton, P.J. and Rohila, J.S. 2011. The Potential of Proteomics Technologies for Crop Improvement under Drought Conditions. Critical Reviews in Plant Sciences. 30:471-490.


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: During the first year of this project two Ph.D. students, one visiting scientist and one undergraduate student were trained for wheat transformation and wheat, soybean and rice proteomic technologies. For this period the project was mainly focused on student training, standardizing the protocols in the newly established lab and producing preliminary results. The results of the project were presented at two state level scientific meetings and one international level meeting. PARTICIPANTS: People involved on this project during this period include Ansuman Roy (visiting scientist), Manali Shirke (graduate student), Moustafa Eldakak (graduate student) and Kim Buehner (undergraduate student). TARGET AUDIENCES: Target audiences for this project are plant biology students, researchers in the US and around the globe. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
During this first year of the project we have standardized the wheat transformation and proteomics protocols. Towards the goal of understanding the drought tolerance phenomenon we have performed a series of drought experiments in wheat and soybean plants. By performing proteomic studies we have found a number of differentially expressed protein spots on 2D gel electrophoresis as a result of regulated drought stress in wheat and soybean plants. The mass spectrometric identification of the novel proteins is underway. The findings of novel drought resistance genes will add immensely to our understanding of this crop yield reducing stress at molecular levels.

Publications

  • 1. Roy, A. and Rohila, J. S. 2010. 2-DE profiling of total proteome changes in cereals and oil crops under abiotic stresses. In: 95th Annual Meeting of South Dakota Academy of Science. 2010. Spearfish, SD, USA.
  • 2. Shirke, M., Buehner, K., Roy, A., Rohila, S. and Rohila, J.S. 2010. Establishment of an efficient callus induction and regeneration protocol from immature embryos of wheat. In: 95th Annual Meeting of South Dakota Academy of Science, April 9-10, 2010. Spearfish, SD, USA.
  • 3. Liu, Z., Rohila, J. S., Liu, W., Wang, Q., and Yang, Y. 2010. A Nep1-like Protein Toxin from Magnaporthe oryzae Interacts with a Conserved, Ubiquitin-like Rice Protein and Elicits Plant Cell Death. In: The 5th International Rice Blast Conference, August 12-14, 2010. Little Rock, Arkansas, USA.