An engineered promoter driving expression of a microbial avirulence gene confers recognition of TAL effectors and reduces growth of diverse Xanthomonas strains in citrus

Abstract

Xanthomonas citri ssp. citri (X. citri), causal agent of citrus canker, uses transcription activator-like effectors (TALEs) as major pathogenicity factors. TALEs, which are delivered into plant cells through the type III secretion system (T3SS), interact with effector binding elements (EBEs) in host genomes to activate the expression of downstream susceptibility genes to promote disease. Predictably, TALEs bind EBEs in host promoters via known combinations of TALE amino acids to DNA bases, known as the TALE code. We introduced 14 EBEs, matching distinct X. citri TALEs, into the promoter of the pepper Bs3 gene (ProBs3 ), and fused this engineered promoter with multiple EBEs (ProBs3 ) to either the β-glucuronidase (GUS) reporter gene or the coding sequence (cds) of the pepper gene, Bs3. TALE-induced expression of the Bs3 cds in citrus leaves resulted in no visible hypersensitive response (HR). Therefore, we utilized a different approach in which ProBs3 and ProBs3 were fused to the Xanthomonas gene, avrGf1, which encodes a bacterial effector that elicits an HR in grapefruit and sweet orange. We demonstrated, in transient assays, that activation of ProBs3 by X. citri TALEs is T3SS dependent, and that the expression of AvrGf1 triggers HR and correlates with reduced bacterial growth. We further demonstrated that all tested virulent X. citri strains from diverse geographical locations activate ProBs3 . TALEs are essential for the virulence of X. citri strains and, because the engineered promoter traps are activated by multiple TALEs, this concept has the potential to confer broad-spectrum, durable resistance to citrus canker in stably transformed plants.

Mechanistic insights into host adaptation, virulence and epidemiology of the phytopathogenXanthomonas

“…Zeng et al engineered the promoter region of Xa10 executor R gene to contain EBE recognition site of multiple X. oryzae TALEs and demonstrated that the modified executor R gene, Xa10 (E5), provided broad spectrum resistance against 27 X. oryzae field isolated (Zeng et al 2015). Using a similar approach, Shantharaj and colleagues introduced the EBE region of the CsLOB1, the canker susceptibility gene induced by X. citri PthA4, into the promoter of the Bs3 gene and replaced Bs3 with avrGf1 which is recognised as an avirulence gene in grapefruit (Shantharaj et al 2017). Transient expression of the engineered executor R gene in grapefruit leaves leads to the development of HR in response to X. citri and reduced bacterial colonisation (Shantharaj et al 2017).…”

“…Using a similar approach, Shantharaj and colleagues introduced the EBE region of the CsLOB1, the canker susceptibility gene induced by X. citri PthA4, into the promoter of the Bs3 gene and replaced Bs3 with avrGf1 which is recognised as an avirulence gene in grapefruit (Shantharaj et al 2017). Transient expression of the engineered executor R gene in grapefruit leaves leads to the development of HR in response to X. citri and reduced bacterial colonisation (Shantharaj et al 2017).…”

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

“…Engineering of plant R genes through rational design relies on well-characterized functional modules. TALE-inducible plant R promoters are well understood, which allows for the design of R promoters that are capable of recognizing multiple TALEs instead of the single-TALE recognition of native R promoters (Hummel et al, 2012;Strauß et al, 2012;Shantharaj et al, 2017). In contrast to our detailed understanding of TALE-specific R promoters, the molecular basis of executor R protein triggered defense reactions remains mostly obscure.…”

PthA4AT, a 7.5‐repeats transcription activator‐like (TAL) effector from Xanthomonas citri ssp. citri, triggers citrus canker resistance

“…They trigger host responses associated with HR, similarly to classical R genes like receptor like‐kinases (RLK) and nucleotide binding site‐leucine‐rich repeat (NBS‐LRR) genes, but if the resistance pathways triggered by the TALE‐mediated executor R genes intersect with those of the other R genes remain unknown. Agrobacterium ‐mediated delivery of a 35S promoter‐driven executor Bs3 coding‐region did not cause a visible HR in citrus (Shantharaj et al ., ). The functionality of the other known executor R proteins in citrus is unknown.…”