Source: CLEMSON UNIVERSITY submitted to
GENETIC ENGINEERING FOR SEPTORIA DISEASE RESISTANCE IN HYBRID POPLAR
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0224884
Grant No.
2011-67010-30197
Project No.
SCW-2010-04180
Proposal No.
2010-04180
Multistate No.
(N/A)
Program Code
A6121
Project Start Date
Mar 15, 2011
Project End Date
Mar 14, 2014
Grant Year
2011
Project Director
Liang, H. L.
Recipient Organization
CLEMSON UNIVERSITY
(N/A)
CLEMSON,SC 29634
Performing Department
(N/A)
Non Technical Summary
Septoria musiva, one of the most damaging pathogen in Populus spp., causes leaf spot and canker diseases and is directly responsible for plantation failure. Chemical and cultural control has proven problematic and largely ineffective. To date, the planting of resistant clones appears to the best means of controlling this pathogen. This project aims to generate and test transgenic hybrid poplar plants that carry candidate disease resistance genes and to identify key genes that are involved in the response to S. musiva infection. The resistance of the transgenic plants to S. musiva will be evaluated by in vitro leaf disc assays and compared to the untransformed control. Gene expression profiling between resistant and susceptible clones will be compared to identify candidate genes that can be useful in conferring disease resistance in poplars and other species. Spatial expression pattern of the key candidate genes identified in microarray analyses will be determined by quantitative RT-PCR. This study will help identify new opportunities for improving and developing poplar clones that are resistant to Septoria leaf spot and canker diseases, therefore improve plantation productivity. The information obtained here will also provide data necessary to seek funding for further study of developing and improving Septoria-resistant poplar clones.
Animal Health Component
(N/A)
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1250670104010%
1252499104010%
2010670104010%
2012499104020%
2030670104010%
2032499104010%
2120670104020%
2122499104010%
Knowledge Area
125 - Agroforestry; 203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 212 - Pathogens and Nematodes Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
0670 - Short rotation woody crops, including holiday trees; 2499 - Plant research, general;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
The overall goals of this project is to generate and test transgenic hybrid poplar plants that carry candidate disease resistance genes and identify key genes that are involved in the response to S. musiva infection. Specific objectives include: 1)Generation of transgenic hybrid poplar plants for the six constructs carrying either a sole wheat oxalate oxidase gene, a sole ESF39 antimicrobial peptide gene (pTACF6), a sole Chinese chestnut laccase gene (pESF-KBL), or in combination with wheat oxalate oxidase gene plus the Chinese chestnut laccase (pESF-KBLO), wheat OxO gene plus ESF39 antimicrobial peptide gene (pTACF7), and a triple pyramid with all three genes (pESF-KBLOE). 2)Leaf disc assays to score the resistance of the transgenic plants to S. musiva in comparison to the untransformed control. The transgenic poplars that show resistance will be sent to collaborator Dr. Glen Stanosz in Wisconsin for future canker and leaf spot disease tests in the field. 3)Microarray analysis to identify genes responsive to Septoria infection. Expression profiling from the wild-type susceptible and resistant clones will be compared. 4)Quantitative RT-PCR (reverse transcription-polymerase chain reaction) analysis to confirm the major candidate genes that are responsive to S. musiva infection and to determine these genes' spatial expression patterns. The expected outputs include transgenic plants with S. musiva resistance, genes that are involved in poplar/S. musiva interactions, publications, and training of a postdoctoral fellow.
Project Methods
Poplar transformation, regeneration, and acclimation: Agrobacterium-mediated transformation will be conducted on a S. musiva highly susceptible clone, OGY (P. deltoides x P. nigra), with six binary vectors. Selection will be carried out with kanamycin. At least 10 transgenic plants representing different transformation events per binary construct will be generated. After PCR screening, the transgenic plants will be elongated and rooted with kanamycin in Magenta cubes. The transgenic and untransformed plants are then acclimated in potting mix in a gradually vented-growth chamber for three weeks before transferred to a greenhouse facility. All the transgenic lines will be further validated by Southern hybridization and transgene expression will be examined by quantitative RT-PCR and related to their resistance to S. musiva. In vitro leaf disc assay with S. musiva: To evaluate the S. musiva resistance in the transgenic poplar plants, in vitro leaf disk assays will be performed. Briefly, S. musiva conidia will be collected and used to inoculate transformed and untransformed hybrid poplar OGY leaf disks. After two weeks of incubation at 22C under a 16hr photoperiod of cool-light fluorescent light, percentage of necrotic area from transgenic and untransformed OGY will scored by the NIH Image 1.63 program. Fifteen leaf disks from each transformed line and the untransformed control will be included in each trial and a total of three trials will be preformed. Comparisons among plants carrying different gene constructs and to untransformed control will be conducted by ANOVA. Multiple pairwise comparisons will be based on Tukey's Studentized Ranged Test. Microarray analysis: An updated Agilent microarray will be employed to identify genes responsive to Septoria infection. RNA samples from infected and uninfected leaves and stems of wild-type susceptible and resistant clones will be isolated and used in the microarray experiments. Infected leaves will be collected from 4-month-old cuttings grown in a growth chamber and five days after S. musiva inoculation by spraying. Cankers will be inoculated by removing the leaf at the plastochron index 5 of 5-month-old cuttings and placing a S. musiva agar culture plug with the mycelium side toward the stem. Whole stems within 2 cm radius of the inoculation site will be used for RNA extraction. Three biological and three technical repeats will be included and a two-dye labeling approach will be employed. Raw data will be filtrated and normalized by a nonlinear normalization) method that utilizes gene intensity and spatial information. To select the differentially expressed genes, the concept of false discovery rate will be utilized. Quantitative RT-PCR analysis: Quantitative RT-PCR will be conducted to validate key S. musiva-responsive genes identified in the microarray analysis by using primers designed from 5' and/or 3' unique UTR sequences of the candidate genes. RNA isolated from different tissue types, including xylem, phloem, leaf, petiole, and roots, will be used in the quantitative RT-PCR analyses to determine the spatial expression of genes of interest.

Progress 03/15/11 to 03/14/14

Outputs
Target Audience: The target audiences include high school and undergraduate students, postdoctoral fellows, and professionals from academia, industry, and government. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project has provided research trainings for 2 high school and 4 undergraduate students, as well as trained a postdoctoral fellow. Through seminars, we have increased students’ knowledge of plants and their responses to biotic stresses. How have the results been disseminated to communities of interest? The results been disseminated to communities of interest through publications, conference presentations, and seminars. What do you plan to do during the next reporting period to accomplish the goals? Complete whole-plant inoculation on the transgenic plants and submit a manuscript on the evaluation of effects of the candidate resistance genes.

Impacts
What was accomplished under these goals? Transformation of poplar with 6 constructs was completed. Expression levels of transgenes were examined with qRT-PCR. Inoculation of Septoria musiva in whole plants is being conducted. Identification of poplar genes in response to S. musiva infection via differential expression analysis has been completed in leaf tissues.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Haiying Liang, Margaret Staton, Yi Xu, Tao Xu, Jared LeBoldus, Comparative expression analysis of resistant and susceptible Populus clones inoculated with Septoria musiva. Plant Sci 223: 6978
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Haiying Liang, Margaret Staton, Yi Xu, Tao Xu, Jared LeBoldus Dissecting the molecular mechanisms underpinning the susceptibility of poplar (Populus spp.) to Septoria musiva. Plant Biology Annual Meeting (Providence, RI, July, 2013, (poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Haiying Liang, Margaret Staton, Shivegowda Thammannagowda, Yi Xu, Tao Xu, Jared LeBoldus? "Enhancing Septoria musiva resistance in poplar with molecular genetic approaches"?Oral presentation in and proceedings of South Forest Tree Improvement Conference, Clemson SC, June 10-14
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Y Xu, Shivegowda Thammannagowda, H Liang: Development of Disease Resistant Poplar Trees using Transformation Approaches. American Society of Plant Biologists Annual Meeting, July, Austin, Texas, (poster)


Progress 03/15/12 to 03/14/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Microarray was replaced with RNAseq in teh differential expression analysis. What opportunities for training and professional development has the project provided? a postdoctoral fellow, an undergradaute student, and a high school summer intern were trained in this project. How have the results been disseminated to communities of interest? Progress of the project was presented as a poster in the 2012 American Society of Plant Biologists. What do you plan to do during the next reporting period to accomplish the goals? Complete the pathogen inoculation analysis, submit two manuscripts for review, present the results in two conferences.

Impacts
What was accomplished under these goals? Transformation of poplar with 6 constructs was completed. Expression levels of transgenes were examined with qRT-PCR. Inoculation of Septoria musiva in whole plants are being conducted. Identification of poplar genes in response to S. musiva infection via differential expression analysis has been completed in leaf tissues. A manuscript is in writing on the results from differential expression analysis.

Publications

  • Type: Other Status: Published Year Published: 2012 Citation: (2012) Y Xu*, Shivegowda Thammannagowda*, H Liang: Development of Disease Resistant Poplar Trees using Transformation Approaches. American Society of Plant Biologists Annual Meeting, July, Austin, Texas, (poster)


Progress 03/15/11 to 03/14/12

Outputs
OUTPUTS: In the first year of the project, we have obtained six binary constructs from State University of New York, College of Environmental Science and Forestry (SUNY-ESF) and 10 poplar clones from University of Wisconsin through Material Transfer Agreements. The poplars clones are being grown in greenhouse and in tissue culture. A poplar clone "OGY" has been successfully transformed with the six binary vectors individually. We have obtained at least 6 transgenic lines for each vector. All the transgenic plants have been rooted and propagated in the greenhouse. The transgene expression analysis by RT-PCR is complete. Transgenic plants and the 10 wildtype clones are being inoculated with Septoria musiva for microarray study and symptom scoring. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This project has helped trained a postdoctoral fellow, one undergraduate student, and a high school student (who is now an undergraduate student in University of South Carolina) in Year One. The project led to a campus visit from a tree pathology who gave an exciting and informative seminar to an audience of approximately 60 students and faculty. Besides the collaboration with SUNY-ESF and University of Wisconsin, we have initiated collaboration with North Dakota State University.

Publications

  • No publications reported this period