Analyses Of TALE-induced Resistance And Putative Susceptibility Genes In Tomato

Abstract

Transcription activator-like effectors (TALEs) from Xanthomonas spp. interact with effector-binding elements (EBEs) in the promoter regions of their target genes to upregulate host susceptibility (S) genes for virulence enhancement. To counteract the pathogen, pepper and rice species have evolved so-called executor resistance (R) genes, which upon transcriptional activation by TALEs lead to immediate cell death and restrict bacterial growth. Additionally, plants have evolved nucleotide-binding domain leucine rich repeat-containing proteins (NLRs) that are capable of TALE recognition and mediation of cell death. While NLR-mediated immunity pathways are well-studies, executor-mediated pathway components are unknown.

Bacterial spot 3 (Bs3), an executor R gene from pepper, causes cell death upon transcriptional activation by the corresponding TALE from Xanthomonas euvesicatoria, i.e. AvrBs3, while tomato Bacterial spot 4 (Bs4) NLR protein mediates recognition of numerous TALEs. As pepper is not amenable to transformation, the genetic dissection of Bs3-mediated pathways in pepper is not straightforward. Therefore, the main aim of this work was to generate Bs3 transgenic tomato lines lacking Bs4 as a tool to decipher Bs3-mediated pathways.

CRISPR/Cas9-mediated Bs4 mutagenesis yielded null alleles containing mutations within Bs4 coding sequence (CC-Bs4). Phenotyping experiments revealed that tomato lines containing CC-Bs4 alleles no longer showed TALE-dependent cell death. As the next step, transgenic tomato line containing estradiol-inducible Bs3 was generated. Upon transcriptional activation by estradiol in this line, Bs3 led to the cell death suggesting that Bs3-mediated pathway components are conserved between tomato and pepper. In addition, a designer TALE (dTALE) was engineered to bind the estradiol-inducible promoter upstream of the Bs3 CDS. When delivered by X. euvesicatoria, this dTALE activated transcription of the Bs3 transgene and led to a cell death phenotype. Analysis of bacterial growth showed that transcriptional activation of Bs3 correlated with reduced in planta growth of dTALE-containing X. euvesicatoria strain. In summary, the engineered Bs4 knockout line carrying the Bs3 transgene provide a basis for genetic dissection of the Bs3-mediated cell death and immunity pathway.

In addition, it was tested if the knockout of Bs4 affects TALE-dependent host gene activation. Even though Bs4 had no impact on TALE-dependent transcriptional activation of studied host genes, Bs4 was found to be epistatic to TALE-induced disease symptoms.