UC Berkeley

This work provides a comprehensive study of the molecular events mediated by the transcription activator-like (TAL) effectors of Xanthomonas axonopodis pv. manihotis (Xam) during infection of its host plant cassava (Manihot esculenta). TAL effectors are secreted via the bacterial type III secretion system into plant cells where they localize to the nucleus, bind specific sequences of nucleotides in plant promoters, and activate the expression of downstream genes. The DNA binding activity of Xam TAL effectors is carried out by a central domain containing a variable number of 34 amino acid repeats, where the 12th and 13th amino acid residues, the repeat variable diresidues (RVDs), of each repeat dictate which nucleotide will be bound at that site. Genetic analysis of the TAL effectors of highly virulent Xam strain Xam668 showed a virulence role for TAL20Xam668, which contributes to in planta bacterial growth and watersoaking symptom development, and TAL14Xam668, which contributes to in planta bacterial growth. RNA-Sequencing (RNA-Seq) revealed a single host gene target of TAL20Xam668, MeSWEET10a, which is a member of the SWEET family of sugar transporters. Designer TAL effectors that activate MeSWEET10a complemented the Xam668∆TAL20 mutant phenotype, revealing the role of MeSWEET10a in cassava’s susceptibility to Xam. SWEETs are known X. oryzae pv. oryzae TAL effector-targeted susceptibility genes in rice and thus this result establishes the activation of SWEET sugar transporters as a common mechanism of disease promotion for diverse species of Xanthomonas. In contrast to TAL20Xam668 which has a single target gene, RNA-Seq showed that TAL14Xam668 activates over 50 genes during the infection process. A subset of the TAL14Xam668-targeted genes were tested for activation by TAL14CIO151 from Xam strain CIO151 and, although TAL14CIO151 and TAL14Xam668 differ by only a single RVD, they display differential activation of host gene targets. TAL14CIO151 complements the TAL14Xam668 mutant defect, implying that shared target genes are important for TAL14Xam668-mediated disease susceptibility. This type of complementation with closely related TAL effectors is a novel approach to narrowing down biologically relevant susceptibility genes of TAL effectors with multiple targets. The comparison of the activities of TAL14Xam668 and TAL14CIO151 provides an example of how TAL effector target activation by two strains within a single species of Xanthomonas can be dramatically affected by a small change in RVD-nucleotide affinity at a single site and reflects the parameters of RVD-nucleotide interaction determined using artificial TAL effectors in transient systems. Finally, we present proof-of-concept experiments showing TAL effector-induced resistance to Xam in cassava.