Baker Lab Research on Plant Innate Immunity

Our research focuses on understanding molecular-genetic mechanisms of plant innate immunity. We are investigating the structure, function and evolution of host genes for pathogen disease resistance. Our experimental system includes viral, bacterial and oomycete plant pathogens and their Solanaceae plant hosts. We anticipate that our studies will lead to new environmentally benign strategies for durable, broad-spectrum disease resistant crops.

Tobacco mosaic virus (TMV) triggers the N (Necrotic) resistance gene-mediated hypersensitive response (HR, brown lesions).

Structure Function Analysis of the helicase domain of the TMV replicase proteins

(Les Erickson, University of California, Berkeley) Biochemical analyses in vitro analysis of recombinant p50 protein shows it can hydrolyze ATP, as predicted by the presence of ATPase/helicase motifs located in this portion of the replicase proteins. A point mutation in one of these motifs (the P-loop) abolishes ATPase activity, but does not alter cell death-inducing activity, indicating enzymatic activity is not required for elicitation.

NPK1- and SGT1-silenced N. benthamiana Gene Expression Studies by Microarray Analysis

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.

Virus Induced Gene Silencing of Candidate Genes in N. benthamiana

(Hailing Jin, UC Berkeley) 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.

Generation of a Nicotiana benthamiana full-length cDNA database

((in collaboration with TIGR and Brian Staskawicz’s group) Hailing Jin, UC Berkeley) 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.

Characterization of Transgenic SGT1-silenced plants

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.

Biochemistry of N Gene Function

(Vannesa Handley, University of California, Berkeley) Employing Agrobacterium inoculation as a delivery system, we are introducing expression constructs containing various portions of the viral replicase into tobacco. These constructs are then scored for their ability to elicit N-dependent HR at the infiltration site.

MicroRNA regulation of plant innate immune receptors

Plant innate immunity depends on recognition of pathogen effectors and triggering of host defenses. Plant resistance (R)-genes encode a major class of innate immune receptors. Plant microRNAs (miRNAs) and small interfering RNAs (siRNAs) guide gene silencing and play essential regulatory roles in development, genome function and host defense. We discovered novel miRNA families whose members silence R-genes including those encoding resistance to major pathogens of Solanaceae crops.