Progress 09/01/08 to 08/31/10
Outputs
OUTPUTS: The Gastrodia anti-fungal protein (GAFP-1) is a monocot mannose-binding lectin found in the Asiatic orchid Gastrodia elata. Wild-type (WT) plum tissue was budded onto transgenic plum lines 4J and 4I to create chimeric-grafted trees. Tissues from chimeric-grafted trees were analyzed for gafp-1 transcripts (leaf and root) and protein (leaf, soft shoot, and root) by RT-PCR and immunodetection, respectively. Transcripts of gafp-1 were detected consistently in the root tissues but not within the leaves of the grafted, WT scions. Similarly, the GAFP-1 lectin was identified within the roots, but not in the soft shoot or leaf tissues of the grafted, WT scions. These results suggest that gafp-1 mRNA and protein are not moving into the WT scion tissues of chimeric-grafted plum trees. The expression of gastrodianin antifungal protein (GAFP) in a form of its VNF isoform increases tolerance to Phytophthora root rot (Phytophthora cinnamomi) and the root-knot nematode (Meloidogyne incognita) in transgenic plum lines. However, nothing is known about the potential of the GAFP lectin to confer disease resistance to the ring nematode, Mesocriconema xenoplax, in plum. Three transgenic plum lines (4I, 4J, and 5D) expressing gafp-1 under the control of CaMV 35S promoter sequence were evaluated for their response to M. xenoplax in the greenhouse. All plum lines were rated as hosts of M. xenoplax. Among the individual plum lines tested, the number of M. xenoplax per gram of dry roots was lowest in the rhizosphere of transgenic line 5D, intermediate in that of the nontransformed control line, and greatest in line 4J. Eighteen Prunus rootstock cultivars and selections budded with Redhaven peach were planted at 16 locations in North America in 2009. After 2 years, Guardian rootstock was one of the most vigorous and high survival rootstocks in this trial. We used peach cultivars with contrasting chilling requirements (CR) for bud break to observe the expression of DAM3, DAM5 and DAM6 genes in response to chilling accumulation in the field and controlled environments. Vegetative terminal and floral buds were sampled weekly from field grown Contender (1050 hours CR), Rubyprince (850 hours CR) and Springprince (650 hours CR) through winter 2009-2010. Flower and vegetative terminal bud break potential was evaluated at each sampling by forcing cuttings in a growth-permissive environment. We also measured vegetative terminal bud break and DAM gene expression in potted Contender and Peen-To (450 hours CR) trees under controlled-environment cold exposure. DAM3, DAM5 and DAM6 genes were suppressed by exposure to chilling temperatures in the field and in controlled conditions. Expression of DAM5 and DAM6 are higher in high chill cultivars prior to chilling accumulation and their expression level reaches a minimum in each cultivar coincident with acquisition of bud break competence. Expression levels of DAM5 and DAM6 in vegetative tips in controlled environment conditions were negatively correlated with the time required for bud break in forcing conditions. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Transgenic plum (Prunus domestica var. Stanley) lines (4J and 4I) expressing GAFP-1 exhibit enhanced disease resistance to the stramenopile pathogen Phytophthora cinnamomi and the root-knot nematode Meloidogyne incognita. Rootstocks created from transgenic lines might be more readily accepted by consumers if it can be shown that foreign gene products are not migrating into a grafted, non-transgenic scion on which fruit is produced. The results from the nematode study indicate that the comparisons of the final soil densities (Pf) of adult and juvenile M. xenoplax expressed as nematodes per gram of dry roots provide a better measure of the nematode carrying capacity by the tested lines than Pf values referred to as number of M. xenoplax/100 cm3 soil. Guardian rootstock, which is tolerant to the ring nematode/bacterial canker complex, is performing well across many sites, and its disease tolerance may be largely genetic and not influenced as much by soil and climatic factors. The expression patterns of DAM5 and DAM6 genes are consistent with a role as quantitative repressors of bud break. This information is useful to determine when peach trees exit dormancy and how it impacts a tree's cold hardiness and resistance to bacterial canker.
Publications
- Okie, W.R., G.L. Reighard, and A.P. Nyczepir. 2009. Importance of Scion Cultivar in Peach Tree Short Life. J. American Pomological Society 63(2):58-63.
- Nagel, A. K., H. Kalariya, and G. Schnabel 2010. The Gastrodia antifungal protein (GAFP-1) and its transcripts are absent from scions of chimeric-grafted plum. HortScience 45:188-192.
- Nyczepir, A. P., A. K. Nagel, and G. Schnabel 2009. Host status of three transgenic plum lines to Mesocriconema xenoplax. HortScience 44:1932-1935.
- Jimenez S., Reighard G.L., Bielenberg D.G. 2010. Gene expression of DAM5 and DAM6 is suppressed by chilling temperatures and inversely correlates with bud break rate. Plant Molecular Biology 73(1-2): 157-167.
- Jimenez S, Z. Li, G.L. Reighard, and D.G. Bielenberg. 2010. Identification of genes associated with growth cessation and bud dormancy release using a dormancy-incapable tree mutant. BMC Plant Biology 10(25).
- Fan, F., D. G. Bielenberg, T.N. Zhebentyayeva, G. L. Reighard, W.R. Okie, D. Holland and A. G. Abbott. 2009. Mapping quantitative trait loci associated with chilling requirement, heat requirement and bloom date in peach (Prunus persica). New Phytologist doi: 10.1111/j.1469-8137.2009.03119.x
- Li, Z., G.L. Reighard, A.G. Abbott and D. G. Bielenberg. 2009. Dormancy-associated MADS genes from the EVG locus of peach [Prunus persica (L.) Batsch] have distinct seasonal and photoperiodic expression patterns. J. Experimental Botany 60 (12): 3521-3530.
- Jimenez, S., A. L. Lawton-Rauh, G. L. Reighard, A. G. Abbott, and D. G. Bielenberg. 2009. Phylogenetic analysis and molecular evolution of the dormancy associated MADS-box genes from peach. BMC Plant Biology 2009, 9:81.
|
Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Agrobacterium tumefaciens mediated transformation yielded 21 gafp-1 expressing plum lines (Prunus domestica var. BlueByrd). These lines are currently propagated, characterized and evaluated for disease resistance. Collaborations with UC Davis were established to test these lines for Armillaria root rot resistance under controlled conditions. Field trials were initiated to determine the potential for trunk infusions to manage Armillaria root rot. Specifically, trees surrounding hot spots were infused in the spring and fall of 2008. Additionally, residual propiconazole in roots infused 6 and 12 months before root harvest was investigated. Preliminary results show that the infusion may have to be done twice a year for effective control. Two more field experiments investigating biological control options and cultural practices were maintained and tree vigor and disease incidence data was collected. So far, not enough trees have died or expressed symptoms to separate treatments.We have completed the screening of the DAM genes expression in response to nutrient and water stresses in actively growing trees. We have recently completed sampling of vegetative and floral bud tissue from three cultivars with a range of chilling requirements from endodormancy establishment weekly through chilling fulfillment. We are currently analyzing DAM gene expression in these tissues to establish whether shorting days or cold accumulation is the major regulator of winter expression of the DAM gene family. One hundred and seventy microsatellite/simple sequence repeat (SSR) markers, each uniquely mapped to chromosomal locations on the Prunus reference genome, were used to screen the two parents and F1-11. Forty-seven SSR markers showed polymorphism among the parents and were heterozygous in F1-11. Segregation data obtained from the F2-11 population for SSR marker inheritance and their PTSL-response in a PTSL field trial were compiled to identify nuclear genomic regions associated with the response to PTSL. Of the 47 polymorphic SSRs, nine (distributed on 4 linkage groups) were genetically linked with the response to PTSL. Using Joinmap 3.0 software, a genetic map with seven linkage groups was constructed from 30 of the 47 polymorphic markers with coverage of 217.5 cM. QTL analysis was conducted using PLABQTL1.2 software by implementing the interval mapping method. QTLs were detected from 0 to 10 cM from the upper terminus of linkage group 2. 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 research will result in an integrated strategy to manage ARR disease. Components of the strategy are being tested individually. The results of our studies suggest that the expression of gafp-1 in the roots of a woody plant confers some level of resistance to PRR and root-knot nematode. Lines with a stronger promoter may provide even more disease resistance. Greenhouse and long term field trials are underway to confirm this hypothesis. It is still too early to tell whether our ARR field experiments can manage this disease effectively. Two SSR markers located within the QTL of the Prunus reference genome were also found to be associated with the response to PTSL by individual marker analysis. The upper region of linkage group 2 appeared to be important because both the individual SSR analysis and the QTL analysis linked this region with the response to PTSL. The genes controlling tolerance or susceptibility to PTSL may reside in this region.
Publications
- Amiri, A., Bussey, K. E., Riley, M. B., Schnabel, G. 2008. Propiconazole inhibits Armillaria tabescens in vitro and translocates into peach roots following trunk infusion. Plant Dis. 92:1293-1298. Nagel, A.K., Schnabel, G., Petri, C. and Scorza, R. 2008. Generation and characterization of transgenic plum lines expressing the Gastrodia antifungal protein. HortScience 43:1514-1521.
- Zhebentyayeva, T.N., G. Swire-Clark, L. Georgi, L. Garay, S, Jung, S. Forrest, A.V. Blenda, B. Blackmon, J. Mook, R. Horn, W. Howad, P. Arus, D. Main, J.P .Tomkins, B. Sosinski, W.V. Baird, G.L. Reighard and A.G. Abbott. 2008. A framework physical map for peach, a model Rosaceae species. Tree Genetics and Genomes DOI: 10.1007/s11295-008-0147-z.
- Reighard, G.L., D. R. Ouellette, and K. H. Brock. 2008. Performance of new Prunus rootstocks for peach in South Carolina. Acta Hort. 772:237-240.
- Amiri, A., K. E. Bussey, M. B. Riley, and G. Schnabel 2008. Basipetal translocation of propiconazole trunk infusion of peach trees. Phytopathology 98:S12.
- Bielenberg D.G., Y. Wang, Z. Li, T. Zhebentyayeva, S. Fan, G.L. Reighard, R. Scorza, and A.G. Abbott. 2008. Sequencing and annotation of the evergrowing locus in peach [Prunus persica (L.) Batsch] reveals a cluster of six MADS-box transcription factors as candidate genes for regulation of terminal bud formation. Tree Genomes & Genetics 4:495-507.
|
|