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Functional Genomics of Solanaceae Genome Hotspots for Pathogen Resistance |
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» Research » Functional Genomics of Solanaceae |
The aim is to identify candidate resistance signaling genes, identify TMV defense signaling pathways in Solanaceae, and elucidate the role of son1 in R-gene mediated resistance and basal defense. Microarray analyses of tobacco mosaic virus (TMV)-challenged tomato were initiated to compare global gene expression patterns of wild-type (N) TMV-resistant, wild-type (n) TMV-susceptible, and enhanced susceptibility mutant, son1. Availability of enhanced susceptibility mutant, son1, wild-type resistant (N) and wild-type susceptible (n) tomato, offers a unique opportunity to establish TMV-induced gene expression profiles of resistant and susceptible genotypes differing by a single gene. Gene expression profiles of wild-type TMV-resistant tomato (N), wild-type TMV-susceptible tomato (n), and enhanced susceptibility mutant son1 challenged with TMV and compared to mock treatment were assessed after one hour post-inoculation. Microarray studies underway include comparing gene expression profiles of wild-type resistant, wild-type susceptible and son1 mutant at 3, 9, 27 hours after TMV-challenge and mock treatment.Updated: 08/14/04
The focus of current efforts is on developing an efficient silencing system in tuber-generated potato in order to study the function of candidate genes responsible for certain disease resistance and its signaling pathways. S. bulbocastanum PT29, a wild diploid potato species, is highly resistant to all known races of P. infestans. This broad resistance phenotype is determined by a CC-NBS-LRR type of resistance gene, RB (Song et al., 2003). S. bulbocastanum is sexually incompatible and can only be propagated through tuber vegetative growth making it a difficult host to perform genetic studies. VIGS provides a powerful alternative approach to study gene function. The PDS gene in tuber-generated S. bulbocastanum by inoculating TRV-PDS sap propagated from N. benthamiana resulting in 60-70% of the silenced plants exhibiting a PDS silenced photo-bleached phenotype (Figure 1b). Using the same system, the RB gene in PT29 was silenced.
P. infestans strains, US1 and ME980085, were used to infect RB-silenced PT29 resulting in visible disease symptoms and the loss of resistance (Figure 2). S. tuberosum cultivar Katahdin is susceptible to all the P. infestans strains and was used as a susceptible control. PT29 and TRV-R1 treated PT29 were used as the resistance controls. Four individual experiments with a total of 28 tuber-generated PT29 were used for RB silencing and of those, seventeen silenced-PT29s showed susceptible diseased symptom in the pathogen assay. | | Figure 1. Virus-induced gene silencing of PDS. A) Seed-geminated S. bulbocastanum. B) Tuber-generated S. bulbocastanum. C) Close up of PDS-silenced region of tuber-generated S. bulbocastanum. |
Updated: 08/14/04
Potato cDNA microarrays produced by TIGR were demonstrated to be able to cross-hybridize with N. benthamiana mRNA. It provided a powerful tool for gene expression profiling analysis on VIGS N. benthamiana plants. Gene expression profiles of NPK1- and SGT1- silenced plants were analyzed by using 5K cDNA arrays with biological triplicates. A list of differentially expressed genes was obtained and subjected to further analysis. Several genes have already been analyzed by VIGS. Experiments will be repeated using 10K arrays to analyze expression profiling on NPK1- and SGT1-silenced plants before and after TMV challenge, in order to gain more significant and informative data. | | Selected genes displaying reproducible 2x or greater change in 5K microarray comparisons of silenced N. benthamiana. A) NPK1-silenced vs empty vector treated B) SGT1-silenced vs empty vector treated. |
Updated: 05/18/04
A total of 55 candidate resistance and resistance pathway genes were subjected to functional characterization using virus-induced gene silencing (VIGS). Forty-five candidate genes were chosen from literature searches that may play a role in plant disease resistance and plant-microbial interaction. Six genes were chosen from the list of differentially expressed genes from microarray analysis on NPK1- and Sgt1-silenced N. benthamiana. Four genes were selected from the sequence information of potato BAC clones. Solanaceae N. benthamiana was used for VIGS analysis. Candidate genes were selected from data reported using Arabidopsis, tomato, potato, tobacco, yeast and animals.
Updated: 05/18/04
Nicotiana benthamiana is the best solanaceous plant for performing functional studies of genes via VIGS. A collaboration with Barbara Baker, Brian Staskawicz and Robin Buell at TIGR has been formed to create a Nicotiana benthamiana database, to be added to the public resources for solanaceous plants. The growing public database will ultimately enable parallel analysis of gene functions between Nicotiana benthamiana, potato, tomato and other solanaceous plants.
RNA was isolated from Nicotiana benthamiana tissues that include callus, roots from liquid culture grown plants, heat-stressed leaves (38 C, 3 hr and 6 hr), cold-stressed leaves (5 C 3 hr, 6hr), and pathogen challenged leaves (Pseudomonas syringae pv tomato 12hr; Xanthomonas campestris pv campestris 12 hr, 18hr; Pseudomonas syringae pv phaseolicola 18hr, and Xanthomonas campestris, pv vesicatoria 18hr). RNA was isolated from these tissues and pooled in approximately equal molar amounts. The pooled RNA was used for constructing the normalized library. The gateway based normalized Nicotiana benthamiana cDNA library has been created and ten thousand clones have been sequenced from both 5’ end and 3’ end. All sequence information has been submitted to Genbank.
Updated: 05/18/04
SGT1 plays an important role in almost all R gene mediated disease resistance pathways and some non-host disease resistance pathways (Jin et al., 2002; Peart et al., 2002). In order to investigate the possible role of SGT1 in non-host resistance of tobacco plants to P. infestans, we generated transgenic SGT1-silenced tobacco plants (Samsun NN) using RNAi. Twelve transgenic lines with less severe SGT1 silencing phenotype were recovered. Six out of twelve transgenic lines showed clear attenuation of N mediated TMV resistance pathway. Our results showed that the SGT1-silenced tobacco plants were resistant to the three strains of P. infestans, which leads to the conclusion that SGT1 is not involved in the non-host resistance to P. infestans in tobacco or the transgenic plants were only partially silenced and the remaining SGT1 mRNA may be sufficient for resistance to P. infestans.Updated: 05/18/04
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» Research » Functional Genomics of Solanaceae |
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