RNA-Seq – an alternative to cloning

PNASTranscription activator-like effector (TALE) proteins of the plant pathogenic bacterial genus Xanthomonas bind to and transcriptionally activate host susceptibility genes, promoting disease. Plant immune systems have taken advantage of this mechanism by evolving TALE binding sites upstream of resistance (R) genes. For example, the pepper Bs3 and rice Xa27 genes are hypersensitive reaction plant R genes that are transcriptionally activated by corresponding TALEs. Both R genes have a hallmark expression pattern in which their transcripts are detectable only in the presence and not the absence of the corresponding TALE.

Now a multinational team led by researchers at Ludwig-Maximilians-University have used transcriptome profiling (RNA-seq) in an attempt to avoid laborious positional cloning for the isolation of TALE-induced R genes. In a proof-of-principle experiment, RNA-seq was used to identify a candidate for Bs4C, an R gene from pepper that mediates recognition of the Xanthomonas TALE protein AvrBs4. They identified one major Bs4C candidate transcript by RNA-seq that was expressed exclusively in the presence of AvrBs4. Complementation studies confirmed that the candidate corresponds to the Bs4C gene and that an AvrBs4 binding site in the Bs4C promoter directs its transcriptional activation. Comparison of Bs4C with a nonfunctional allele that is unable to recognize AvrBs4 revealed a 2-bp polymorphism within the TALE binding site of the Bs4C promoter. Bs4C encodes a structurally unique R protein and Bs4C-like genes that are present in many solanaceous genomes seem to be as tightly regulated as pepper Bs4C. These findings demonstrate that TALE-specific R genes can be cloned from large-genome crops with a highly efficient RNA-seq approach.

  • Strauß T, van Poecke RM, Strauß A, Römer P, Minsavage GV, Singh S, Wolf C, Strauß A, Kim S, Lee HA, Yeom SI, Parniske M, Stall RE, Jones JB, Choi D, Prins M, Lahaye T. (2012) RNA-seq pinpoints a Xanthomonas TAL-effector activated resistance gene in a large-crop genome. Proc Natl Acad Sci [Epub ahead of print]. [abstract]