Cell Death Triggered by the YUCCA-like Bs3 Protein Coincides with Accumulation of Salicylic Acid and Pipecolic Acid But Not of Indole-3-Acetic Acid.

The pepper (Capsicum annuum) resistance gene bacterial spot3 (Bs3) is transcriptionally activated by the matching Xanthomonas euvesicatoria transcription-activator-like effector (TALE) AvrBs3. AvrBs3-induced Bs3 expression triggers a rapid and local cell death reaction, the hypersensitive response (HR). Bs3 is most closely related to plant flavin monooxygenases of the YUCCA (YUC) family, which catalyze the final step in auxin biosynthesis. Targeted mutagenesis of predicted NADPH- and FAD-cofactor sites resulted in Bs3 derivatives that no longer trigger HR, thereby suggesting that the enzymatic activity of Bs3 is crucial to Bs3-triggered HR. Domain swap experiments between pepper Bs3 and Arabidopsis (Arabidopsis thaliana) YUC8 uncovered functionally exchangeable and functionally distinct regions in both proteins, which is in agreement with a model whereby Bs3 evolved from an ancestral YUC gene. Mass spectrometric measurements revealed that expression of YUCs, but not expression of Bs3, coincides with an increase in auxin levels, suggesting that Bs3 and YUCs, despite their sequence similarity, catalyze distinct enzymatic reactions. Finally, we found that expression of Bs3 coincides with increased levels of the salicylic acid and pipecolic acid, two compounds that are involved in systemic acquired resistance.

RNA-seq pinpoints a Xanthomonas TAL-effector activated resistance gene in a large-crop genome.

Transcription 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. By transcriptome profiling using next-generation sequencing (RNA-seq), we tested whether we could 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. We 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.

Promoter elements of rice susceptibility genes are bound and activated by specific TAL effectors from the bacterial blight pathogen, Xanthomonas oryzae pv. oryzae.

*Plant pathogenic bacteria of the genus Xanthomonas inject transcription activator-like effector (TALe) proteins that bind to and activate host promoters, thereby promoting disease or inducing plant defense. TALes bind to corresponding UPT (up-regulated by TALe) promoter boxes via tandemly arranged 34/35-amino acid repeats. Recent studies uncovered the TALe code in which two amino acid residues of each repeat define specific pairing to UPT boxes. *Here we employed the TALe code to predict potential UPT boxes in TALe-induced host promoters and analyzed these via beta-glucuronidase (GUS) reporter and electrophoretic mobility shift assays (EMSA). *We demonstrate that the Xa13, OsTFX1 and Os11N3 promoters from rice are induced directly by the Xanthomonas oryzae pv. oryzae TALes PthXo1, PthXo6 and AvrXa7, respectively. We identified and functionally validated a UPT box in the corresponding rice target promoter for each TALe and show that box mutations suppress TALe-mediated promoter activation. Finally, EMSA demonstrate that code-predicted UPT boxes interact specifically with corresponding TALes. *Our findings show that variations in the UPT boxes of different rice accessions correlate with susceptibility or resistance of these accessions to the bacterial blight pathogen.

Recognition of AvrBs3-like proteins is mediated by specific binding to promoters of matching pepper Bs3 alleles.

The pepper (Capsicum annuum) bacterial spot (Bs) resistance gene Bs3 and its allelic variant Bs3-E mediate recognition of the Xanthomonas campestris pv vesicatoria type III effector protein AvrBs3 and its deletion derivative AvrBs3Deltarep16. Recognition specificity resides in the Bs3 and Bs3-E promoters and is determined by a defined promoter region, the UPA (for up-regulated by AvrBs3) box. Using site-directed mutagenesis, we defined the exact boundaries of the UPA(AvrBs3) box of the Bs3 promoter and the UPA(AvrBs3Deltarep16) box of the Bs3-E promoter and show that both boxes overlap by at least 11 nucleotides. Despite partial sequence identity, the UPA(AvrBs3) box and the UPA(AvrBs3Deltarep16) box were bound specifically by the corresponding AvrBs3 and AvrBs3Deltarep16 proteins, respectively, suggesting that selective promoter binding of AvrBs3-like proteins is the basis for promoter activation specificity. We also demonstrate that the UPA(AvrBs3) box retains its functionality at different positions within the pepper Bs3 promoter and confers AvrBs3 inducibility in a novel promoter context. Notably, the transfer of the UPA(AvrBs3) box to different promoter locations is always correlated with a new transcriptional start site. The analysis of naturally occurring Bs3 alleles revealed many pepper accessions that encode a nonfunctional Bs3 variant. These accessions showed no apparent abnormalities, supporting the supposition that Bs3 functions only in disease resistance and not in other developmental or physiological processes.

Plant pathogen recognition mediated by promoter activation of the pepper Bs3 resistance gene.

Plant disease resistance (R) proteins recognize matching pathogen avirulence proteins. Alleles of the pepper R gene Bs3 mediate recognition of the Xanthomonas campestris pv. vesicatoria (Xcv) type III effector protein AvrBs3 and its deletion derivative AvrBs3Deltarep16. Pepper Bs3 and its allelic variant Bs3-E encode flavin monooxygenases with a previously unknown structure and are transcriptionally activated by the Xcv effector proteins AvrBs3 and AvrBs3Deltarep16, respectively. We found that recognition specificity resides in the Bs3 and Bs3-E promoters and is determined by binding of AvrBs3 or AvrBs3Deltarep16 to a defined promoter region. Our data suggest a recognition mechanism in which the Avr protein binds and activates the promoter of the cognate R gene.