TAL effectors: finding plant genes for disease and defense.

Transcription activator like effectors (TALEs) are injected via the type III secretion pathway of many plant pathogenic Xanthomonas spp. into plant cells where they contribute to disease or trigger resistance by binding to DNA and turning on TALE-specific host genes. Advances in our understanding of TALEs and their targets have yielded new models for pathogen recognition and defense. Similarly, we have gained insight into plant molecules and processes that can be co-opted to promote infection. Recent elucidation of the basis for specificity in DNA binding by TALEs expedites further discovery and opens the door to biotechnological applications. This article reviews the most significant findings in TALE research, with a focus on recent advances, and discusses future prospects including pressing questions yet to be answered. Introduction Transcription activator like effectors (TALEs) are a structurally and functionally distinct class of proteins from plant pathogenic bacteria that are injected via the type III secretion pathway into plant cells. Once in the plant cell, they enter the nucleus, bind to TALE-specific DNA sequences, and turn on the downstream genes. Interaction with plants has selected TALEs that activate host genes that facilitate bacterial colonization and spread. Disease pressure has in turn selected plant adaptations that mount defense in response to TALEs. Thus, TALEs can be virulence factors, plant-recognized 'avirulence' factors, or both. Our understanding of TALEs has been shaped by 20 years of research, beginning with the discovery of the AvrBs3 protein of the pepper and tomato pathogen Xanthomonas campestris pv. vesicatoria (Xcv) Distribution and structural features of TALEs TALEs are found in several but not all pathogenic members of the genus Xanthomonas. Individual Xanthomonas strains may have as few as one or as many as several dozen TALEs TALEs have in common an N-terminus required for type III secretion and a C-terminus containing nuclear localization signals (NLS) and an acidic activation domain (AAD) typical of transcription factors TALEs as transcriptional activators-a historical view The TALE AAD was first shown to be functional in the AvrXa10 protein of the rice pathogen X. oryzae pv. oryzae . This was a novel paradigm for effector triggered immunity, and the first example of what we refer to as an 'executor' R gene, the product of which is not involved in recognition of the effector, but only in carrying out defense when it is expressed. The following year, transcriptional activation of the rice gene Os8N3 by the major virulence factor PthXo1 of Xoo was shown to be important for bacterial multiplication and disease development, giving rise to the notion of host susceptibility (S) genes as targets of TALEs [18 ]. That activation is direct and depends on specific, repeat-mediated binding to the target gene promoter, that is, TALEs are indeed transcription factors, was then demonstrated with AvrBs3 and two of the genes it activates in pepper, the executor gene Bs3 and the putative S TAL effectors Bogdanove, Schornack and Lahaye 395 www.sciencedirect.com Current Opinion in Plant Biology 2010, 13:394-401 gene UPA20 [19 ,20 ]. More recently, the precise nucleotide sequences required for activation of these promoters by AvrBs3 were determined [21 ,22 ]. Highly conserved among genes upregulated by AvrBs3, this UPA box functioned from wherever it was placed in a promoter, directing initiation of transcription from 40 to 50 bp downstream of its 5 0 end [22 ]. Resistance mechanisms that target TALEs Surprisingly diverse R genes have been isolated that mediate resistance to TALE containing xanthomonads In addition to R genes that recognize TALE structure (Bs4) or subvert TALE function (Bs3 and Xa27), which are genetically dominant, there are recessive R genes that interfere with TALE activity. For example, the rice xa13 gene comprises alleles of the S gene Os8N3 (synonym: Xa13) that are not activated by the corresponding TALE PthXo1. These are distinguished by differences in their promoters [18 ,31 ,32]. A recent study showed that the Os8N3 promoter contains a functional UPT PthXo1 box, while all xa13 genotypes do not [33 ]. While mutations in the UPT box of an S gene like Os8N3 may block the action of a single TALE, the rice xa5 gene is postulated to interfere with the function of multiple TALEs. Located on chromosome 5, xa5 encodes a V39E substitution variant of the g subunit of the general transcription factor TFIIA [34]. Notably, transcriptional activation of the rice Xa27 gene by the matching TALE AvrXa27 is severely attenuated in xa5 genotypes [35 ]. Reduced transcriptional activation in xa5 genotypes was also reported for the rice Os11N3 gene targeted by TALE AvrXa7 (G. Antony et al., abstract in Phytopathology 2009, 99:S5). Although complementation was not carried out to show conclusively that the effect was due to xa5, given that TFIIAg is a component of the RNA II polymerase complex, it is tempting to speculate that TALEs recruit the complex via interaction with TFIIAg to activate transcription. The xa5-encoded V39E substitution might reduce the affinity of this interaction, resulting in reduced TALE mediated activation of host target genes important for infection. Intriguingly, TALE PthXo7, from a strain virulent to xa5 genotypes, transcriptionally activates a TFIIAg paralog that is located on chromosome 1 and is normally expressed at very low levels [36 ]. This strain might have evolved PthXo7 as a means of compensating for the reduced functionality of the xa5-encoded protein. In support of this hypothesis, a strain against which xa5 is normally effective reached almost 10-fold higher titers in rice xa5 genotypes when transformed with PthXo7 [36 ]. The variety of the resistance mechanisms plants have evolved to defend against pathogens expressing TALEs is remarkable. This is particularly true in light of the fact that effector triggered immunity in most studied cases is mediated by NB-LRR type R proteins, which function as sentinels TALE targeted genes in disease Host whole genome expression analyses led to the identification of the rice S gene Os8N3 activated by PthXo1 and of UPA20 and several other pepper genes upregulated by AvrBs3 [18 ,20 ,21 ]. Os8N3 is related to MtN3, a Medicago truncatula gene differentially expressed during nodulation [37]. In rice, Os8N3 is required for normal pollen development As noted, Xoo TALE PthXo7 activates TFIIAg1, and some evidence suggests that this contributes to susceptibility in a xa5 background [36 ]. Another rice transcription factor gene, TFX1, a member of the basic region leucine zipper (bZIP) family, is activated by Xoo TALE PthXo6 [36 ]. TFX1 was activated by each of a diverse collection of Xoo isolates, suggesting that PthXo6 is broadly conserved and important. A knockout in the strain from which it was identified indeed caused a reduction in virulence that could be rescued by ectopic, constitutive expression of TFX1 [36 ]. Thus, TFX1 is another S gene important for bacterial blight of rice. The AvrBs3-induced pepper UPA20 gene encodes a transcription factor of the basic helix-loop-helix type. Its transcriptional activation results in the hypertrophy that takes place at infection loci in bacterial spot of pepper [20 ]. Hypertrophy may play a role in eruption of the bacteria onto the leaf surface [38]. Other UPA genes form a diverse group for which roles in susceptibility are still unclear, but intriguingly UPA16 is a member of the MtN3 family, like Os8N3 and Os11N3 [20 ,21 ]. Identification of the UPA genes led to the detection of the UPA box, which played a paramount role in the discovery of the mechanism for DNA recognition by TALEs, discussed below. TALE corresponds directly to the sequence of nucleotides in the binding site, one to one in a linear and contiguous fashion DNA recognition by TALEs Moscou and Bogdanove deciphered the mechanism computationally Boch et al. discovered the code by projecting the RVDs of AvrBs3 onto the UPA box [39 ], which is roughly the same length in base pairs as the number of repeats in AvrBs3 [21 ,22 ], and then using the associations in that alignment to find matches for seven other TALEs in the promoters of their respective activation targets. They then constructed artificial promoter sequences predicted to match three TALEs from X. campestris pv. armoraciae (Xca) and showed that these were functional and specific. Using the Xca TALE Hax3 they also demonstrated that the conserved T at À1 was crucial for promoter activation. They tested the functional significance of the association frequencies for HD, NI, NG, and NS by permutating G, A, T, and C at all locations at which the RVD occurs in the 15 RVD long Xca Hax4 protein (the RVDs occur 3-5 times each). For NS, permutations had no detectable effect. For each of the others, permutation to any but the most frequently associated nucleotide, C for HD, A for NI, and T for NG, abolished detectable activity. In a similar set of experiments, the authors showed that arbitrary, artificial TALEs activated corresponding promoters The most common RVDs and the nucleotides they associate with. Letter height represents frequency relative to other nucleotides for that RVD. Nucleotides represent the plus strand of the binding site, but it is not known whether contacts are made on the plus strand, the minus strand, or both. Frequencies are according to Current Opinion in Plant Biology 2010, 13:394-401 www.sciencedirect.com constructed using the code. With one of them, they tested nucleotide specificity for NN. Consistent with its association frequencies, for NN at four locations in a 13 RVD sequence, G at all positions gave the best activity, A reduced but did not abolish activity, and C and T eliminated detectable activity. The results from both studies indicate that TALE-DNA recognition depends on preferential association of individual RVDs with specific nucleotides. It remains unclear if the mechanism is simple, depending for binding on a minimum number of perfect matches over a certain length alignment, or complex, involving differential contributions of different RVD-nucleotide associations that might depend on their position within the alignment