AbstractMost Xanthomonas species translocate Transcription Activator-Like (TAL) effectors into plant cells where they function like plant transcription factors via a programmable DNA-binding domain. Characterized strains of rice pathogenic X. oryzae pv. oryzae harbor 9-16 different tal effector genes, but the function of only a few of them has been decoded. Using sequencing of entire genomes, we first performed comparative analyses of the complete repertoires of TAL effectors, herein referred to as TALomes, in three Xoo strains forming an African genetic lineage different from Asian Xoo. A phylogenetic analysis of the three TALomes combined with in silico predictions of TAL effector targets showed that African Xoo TALomes are highly conserved, genetically distant from Asian ones, and closely related to TAL effectors from the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Nine clusters of TAL effectors could be identified among the three TALomes, including three showing higher levels of variation in their repeat variable diresidues (RVDs). Detailed analyses of these groups revealed recombination events as a possible source of variation among TAL effector genes. Next, to address contribution to virulence, nine TAL effector genes from the Malian Xoo strain MAI1 and four allelic variants from the Burkinabe Xoo strain BAI3, thus representing most of the TAL effector diversity in African Xoo strains, were expressed in the TAL effector-deficient X. oryzae strain X11-5A for gain-of-function assays. Inoculation of the susceptible rice variety Azucena lead to the discovery of three TAL effectors promoting virulence, including two TAL effectors previously reported to target the susceptibility (S) gene OsSWEET14 and a novel major virulence contributor, TalB. RNA profiling experiments in rice and in silico prediction of EBEs were carried out to identify candidate targets of TalB, revealing OsTFX1, a bZIP transcription factor previously identified as a bacterial blight S gene, and OsERF#123, which encodes a subgroup IXc AP2/ERF transcription factor. Use of designer TAL effectors demonstrated that induction of either gene resulted in greater susceptibility to strain X11-5A. The induction of OsERF#123 by BAI3Δ1, a talB knockout derivative of BAI3, carrying these designer TAL effectors increased virulence of BAI3Δ1 validating OsERF#123 as a new, bacterial blight S gene.Author SummaryThe ability of most Xanthomonas plant pathogenic bacteria to infect their hosts relies on the action of a specific family of proteins called TAL effectors, which are transcriptional activators injected into the plant by the bacteria. TAL effectors enter the plant cell nucleus and bind to the promoters of specific plant genes. Genes that when induced can benefit pathogen multiplication or disease development are called susceptibility (S) genes. Here, we perform a comparative analysis of the TAL effector repertoires of three strains of X. oryzae pv. oryzae, which causes bacterial leaf blight of rice, a major yield constraint in this staple crop. Using sequencing of entire genomes, we compared the large repertoires of TAL effectors in three African Xoo strains which form a genetic lineage distinct from Asian strains. We assessed the individual contribution to pathogen virulence of 13 TAL effector variants represented in the three strains, and identified one that makes a major contribution. By combining host transcriptome profiling and TAL effector binding sites prediction, we identified two targets of this TAL effector that function as S genes, one previously identified, and one, new S gene. We validated the new S gene by functional characterization using designer TAL effectors. Both S genes encode transcription factors and can therefore be considered as susceptibility hubs for pathogen manipulation of the host transcriptome. Our results provide new insights into the diversified strategies underlying the roles of TAL effectors in promoting plant disease.
Efficient enrichment cloning of TAL effector genes fromXanthomonas