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- Materials and Methods
- Supporting Information
Many crops, including rice, wheat, cotton, citrus, tomato, cassava, banana, soybean, sugarcane, and others, suffer losses as a result of infection by pathogenic members of the bacterial genus Xanthomonas. Rice (Oryza sativa), a staple for more than half the world’s population, is host to the xylem pathogen Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight, and the leaf mesophyll pathogen, Xanthomonas oryzae pv. oryzicola (Xoc), which causes bacterial leaf streak. These diseases reduce yields by up to 50 and 30%, respectively (Niño-Liu et al., 2006). Like several Xanthomonas species, Xoo relies on transcription activator-like (TAL) effectors to render the host susceptible to colonization (Bai et al., 2000; Yang & White, 2004; Yang et al., 2006; Sugio et al., 2007; Antony et al., 2010). Xoc also deploys TAL effectors, which, though less well studied, are presumed to contribute similarly to virulence (Makino et al., 2006; Bogdanove et al., 2011).
Named for features shared with eukaryotic transcription factors (Yang et al., 2006), TAL effectors are secreted into host cells via the bacterial type III secretion system (T3SS), then directed to the host cell nucleus by C-terminal nuclear localization signals (Yang & Gabriel, 1995; Van den Ackerveken et al., 1996; Szurek et al., 2001, 2002). There they bind to cognate effector binding elements (EBEs) in specific host gene promoters to activate transcription of those genes using a C-terminal, acidic activation domain (Zhu et al., 1998, 1999; Kay et al., 2007; Römer et al., 2007; Boch et al., 2009; Moscou & Bogdanove, 2009). Binding specificity is dictated by a variable number of central, 33–35 amino acid repeats. In each repeat, a pair of variable residues at positions 12 and 13 (together called the repeat-variable diresidue (RVD)) preferentially associates with a different nucleotide to define the length and sequence of the EBE (Boch et al., 2009; Moscou & Bogdanove, 2009). With this modular protein-DNA recognition mechanism, the pathogen can activate multiple susceptibility (S) genes in the host by deploying different TAL effectors. Several bacterial blight S genes in rice that correspond to Xoo TAL effectors important for virulence have been identified (Yang et al., 2006; Sugio et al., 2007; Antony et al., 2010), and several more S gene candidates for bacterial blight and bacterial leaf streak have been predicted computationally based on RVD sequences of uncharacterized Xoo and Xoc TAL effectors (Moscou & Bogdanove, 2009).
Plants counter TAL effector-wielding pathogens with S gene mimics that cause host cell death and block disease progression when transcriptionally activated. Such normally silent, EBE-controlled ‘executor’ resistance genes (Bogdanove et al., 2010) include the pepper (Capsicum annuum) Bs3 gene, which provides resistance against strains of the bacterial spot pathogen Xanthomonas campestris pv. vesicatoria that express the TAL effector AvrBs3 (Bonas et al., 1989; Römer et al., 2007), and the rice bacterial blight resistance gene Xa27, which is activated by the Xoo TAL effector AvrXa27 (Gu et al., 2005). In addition to executor genes, alleles of major S genes that are immune to activation by the corresponding TAL effector, such as the rice xa13 and xa25 bacterial blight resistance genes, provide another form of defense (Yang et al., 2006; Liu et al., 2011). A third type of resistance directed at TAL effectors, again exemplified by a rice bacterial blight resistance gene, xa5, is a polymorphism in the gamma subunit of general transcription factor TFIIA. A single amino acid substitution in the protein is thought to impair the ability of TAL effectors to recruit the transcriptional machinery to activate target genes (Iyer & McCouch, 2004; Sugio et al., 2007; Gu et al., 2009). Each of these types of resistance, however, is subject to defeat by adaptation of the pathogen TAL effector inventory. For example, executor genes can be defeated by mutation or loss of the corresponding TAL effector, provided the TAL effector is dispensable for virulence, as appears to be the case for AvrXa27 (Tian & Yin, 2009). S gene promoter polymorphisms that confer resistance can be overcome by TAL effectors that target the new sequence or an alternative S gene (Antony et al., 2010). Also, the xa5 gene is rendered ineffective by a strain with two apparent adaptations, a TAL effector that activates the corresponding S gene particularly strongly such that the reduction in activity caused by xa5 might be inconsequential, and a TAL effector that induces expression of a TFIIA gamma paralog that may substitute for the allele found in susceptible plants (Yang et al., 2006; Sugio et al., 2007; B. Yang, unpublished). Furthermore, the latter two types of resistance are genetically recessive, rendering them less easily bred into elite hybrids. A genetically dominant, broad-spectrum, and durable form of resistance effective against pathogens that deploy TAL effectors would be beneficial, but none has been identified.
In an Agrobacterium-mediated transient expression assay in Nicotiana benthamiana, Römer et al. (2009a) showed that amending the Bs3 gene promoter with the AvrXa27 EBE (which they call a ‘UPT box’, which stands for up-regulated by TAL effector) and an EBE matching an AvrBs3 variant called AvrBs3Δrep16 rendered the promoter responsive to all three TAL effectors. This pioneering study suggested that broad-spectrum and potentially durable resistance might be achieved by stable integration of an executor gene engineered in this way to respond to TAL effectors from multiple pathogen strains or even different pathogens.
In previous studies, stable integration of Xa27 into rice under conditions in which it was expressed constitutively resulted in reduced tillering, delayed flowering, and stiff, early-senescing leaves; nonetheless, the expression of the gene indeed conferred resistance to several Xoo strains normally virulent on wildtype Xa27 lines, and partial resistance to a strain of Xoc (Gu et al., 2005; Tian & Yin, 2009). We therefore chose Xa27 to test the notion suggested by Römer et al. (2009a) that functional specificity of an executor gene could be broadened, without deleterious effects associated with constitutive expression, by making its promoter responsive to several distinct TAL effectors.
We added to the Xa27 promoter EBEs corresponding to three additional TAL effectors each from the Xoo strain PXO99A, which harbors AvrXa27, and the Xoc strain BLS256, which does not. Stable integration of this construct into rice produced healthy lines exhibiting gene activation by each of the TAL effectors, and resistance to PXO99A, a PXO99A derivative lacking AvrXa27, and BLS256, as well as two other Xoo and 10 Xoc strains from a diverse collection virulent toward wildtype Xa27 plants. Our results establish the efficacy of executor gene promoter engineering for broader specificity. They also demonstrate that a rice gene for bacterial blight resistance can be readily modified to provide complete protection from bacterial leaf streak as well, a disease for which no major gene resistance in rice has been identified.
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- Materials and Methods
- Supporting Information
A resistance gene that recognizes an effector that otherwise contributes strongly to virulence can be relatively durable owing to the fitness cost to the pathogen of losing or modifying that effector to evade detection (Leach et al., 2001). A striking example is bacterial blight resistance mediated by the rice Xa7 gene, which is triggered by AvrXa7, a major virulence factor in Xoo (Hopkins et al., 1992; Bai et al., 2000; Vera Cruz et al., 2000; Ponciano et al., 2003). However, a mutation that uncouples the virulence and avirulence (resistance-triggering) properties of an effector could result in defeat of the resistance gene. Also, because most resistance genes exhibit pathogen race specificity linked to recognition of a single effector, evolution or introduction of pathogen strains that use alternative effectors for virulence can render a resistance gene ineffective. Against pathogens that rely on TAL effectors for virulence, an executor gene engineered to respond to multiple TAL effectors important for virulence and conserved across a pathogen population could provide durable and broad-spectrum protection.
Here, we established the feasibility of such an approach by adding EBEs to the promoter of the Xa27 gene for resistance to rice bacterial blight and demonstrating consequent broadening of its functional specificity to include Xoo strains virulent toward wildtype Xa27-containing plants as well as strains of the bacterial leaf streak pathogen Xoc, for which no simply inherited resistance genes had been identified in rice. Without altering the coding sequence and by adding < 200 bp of recombinant DNA to the gene promoter, EBE amendment resulted in a single gene with an effective recognition spectrum similar to that achieved by the arduous and time-consuming process of pyramiding multiple resistance genes. Our selection of EBEs was based on available data from one strain each of Xoo and Xoc. Systematic studies to catalog the diversity of TAL effectors in Xanthomonas field populations should enable the rational modification of executor genes to provide broad and durable resistance on a geographically specific basis.
Importantly, each of the targeted TAL effectors individually activated the transgene in the UXO construct. Also, the observed patterns of resistance and susceptibility to Xoo ME1 and Xoc BLS256 across all transgenic lines were as expected based on the corresponding EBE and TAL effector content in each interaction, and they correlated directly with the patterns of Xa27 activation. We used ME1 in addition to PXO99A for the gene expression assays and in place of PXO99A for the virulence assays to distinguish resistance as a result of the EBEs added to Xa27 from resistance mediated by the native AvrXa27 EBE. It should be noted that in addition to its deficiency in avrXa27, ME1 is also disrupted in pthXo6, which neighbors avrXa27; although AvrXa27 makes no measurable contribution to virulence, pthXo6 does (Sugio et al., 2007). The virulence reduction as a result of the disruption of pthXo6 in ME1 is slight, however, and did not mask the resistance conferred by the EBE-amended Xa27 constructs. The disruption of pthXo6 in ME1 did, though, limit the effectors in that strain expected to interact with EBEs in the UXO promoter to PthXo1 and Tal9a. Together, the results strongly suggest that activation of the transgene by the wildtype Xoo and Xoc strains is mediated by at least one, and possibly each, of the corresponding TAL effectors in those strains.
Each of the TAL effectors we chose from PXO99A plays a role in virulence. None of the ones we chose from BLS256 has been characterized with respect to virulence, but each has a well-matched EBE in a putative target gene promoter in rice. The set of EBEs matching these effectors expanded the resistance spectrum of Xa27 to include not only the PXO99A mutant lacking AvrXa27, and BLS256, but two of seven additional, geographically diverse Xoo strains and each of 10 additional, diverse Xoc strains tested. The wide resistance spectrum conferred by these EBEs against the Xoc strains and the narrower spectrum against Xoo strains suggest conservation of one or more of the BLS256 TAL effectors in the Xoc strains, and poor conservation of the PXO99A effectors in that group. This may reflect greater overall TAL effector diversity among Xoo strains, potentially as a result of diversifying selection exerted by the nearly 30 known bacterial blight resistance genes in rice (Niño-Liu et al., 2006). As noted, no simply inherited genes for bacterial leaf streak resistance have been identified in rice (the only identified source of complete resistance is the Rxo1 gene from maize (Zhao et al., 2005)), and this might explain the apparently lesser TAL effector diversity across Xoc strains. To conclude with confidence whether a difference in TAL effector diversity truly exists between Xoo and Xoc, however, a more comprehensive and direct inventory across a broad collection of strains would be necessary.
In light of the absence of major, native genes for resistance to bacterial leaf streak in rice, it is particularly promising that Xa27 was fully effective against Xoc. The only executor gene in rice cloned to date, Xa27 encodes a 113-amino-acid product that has no similarity to functionally characterized proteins, but contains an amino-terminal signal-anchor-like sequence that mediates export to the apoplast and is required for bacterial blight resistance (Wu et al., 2008). Xoo colonizes rice xylem vessels, interacting with cells in the surrounding xylem parenchyma to cause bacterial blight. Xoc multiplies in the mesophyll parenchyma. The mechanism by which Xa27 confers resistance to bacterial blight is unknown. Its activation causes cell death, but it is as yet unclear whether the death is programmed or a result of toxicity of the Xa27 protein. Its effectiveness against bacterial leaf streak, demonstrated here, indicates that the mechanism is general and not restricted to cells in the xylem parenchyma.
As noted earlier, weak constitutive expression of Xa27, though deleterious to the plant, conferred partial resistance to Xoc strain L8 (Tian & Yin, 2009). The complete resistance we observed is likely a result of stronger, localized expression in response to one or more of the targeted TAL effectors delivered by Xoc. Fold induction of Xa27 in the UXO construct by each of the targeted Xoo and Xoc TAL effectors individually correlated positively with proximity of the EBE to a putative TATA box downstream and to the common start site of transcription. Although differences in expression, delivery, or affinity of the TAL effectors may have contributed to this pattern, the strength and significance of the correlation of EBE position to fold induction suggest that there may be a limit to the number of EBEs that can be effectively added to a promoter, as those farthest upstream might drive expression only weakly or not at all. We also observed, unexpectedly, that PXO99A failed to activate Xa27 in the RAN lines, despite its ability to do so in IRBB27. The RAN construct maintains the native AvrXa27 EBE, differing from the UXO construct only in its internal randomization of the added EBEs. Thus, the randomized sequences in some way prevent activation by AvrXa27, or, less likely, in both lines activation is blocked as a result of the positions at which the construct integrated. Although the TAL effector-DNA binding code enables prediction of TAL effector binding sites with relative confidence, these observations highlight the fact that we still know little of the contextual requirements for functional EBEs, with respect to both promoter sequences and chromosomal location and chromatin status. Until more is known, successful placement of EBEs in a promoter may require some trial and error.
Another complexity in EBE amendment is the possibility of interaction among different TAL effectors binding to the same promoter. We sought to minimize this by spacing EBEs 6 bp apart and alternating those for Xoo with those for Xoc. Nonetheless, our 5′ RACE results comparing TSSs detected following individual TAL effector delivery with those detected following inoculation with the corresponding Xoo or Xoc strain provided evidence for such interaction. Some TSSs detected following inoculation with the pathogen strain were not detected on delivery of any of the corresponding individual effectors and, to a lesser extent, vice versa. Thus, although the collection of transcript sequences may not have been saturating, the data suggest that both cooperative interaction to generate novel TSSs as well as interference that blocks initiation from some sites take place.
Previous analyses showed that TAL effector-initiated transcription occurred between 42 and 54 nucleotides downstream of the 3′ end of the EBE, even when the EBE was moved out of context (Kay et al., 2007, 2009; Römer et al., 2007, 2009a, 2009b; Antony et al., 2010). Our results differ. Most transcripts driven by each of the six nonnative TAL effector–EBE interactions on the Xa27 promoter in the UXO construct were initiated at a site shared by all interactions, rather than at sites corresponding to the relative positions of the EBEs. Because this was the same site of transcript initiation observed in mock-inoculated leaves and leaves treated with the nontargeted negative control AvrXa10, it appears that in some configurations, and perhaps depending on the EBE, TAL effector-induced transcript initiation can default to the primary site used for basal expression. The location of the common TSS at 27 bp downstream of a putative TATA box suggests that presence of such an element might influence the location of TAL effector-driven transcript initiation. Some of the TAL effectors reported to dictate the TSS display a TATA box-like sequence within their EBE, which might explain that ability, but not all of them do. Indeed, the AvrXa27 EBE contains no TATA-like sequence, yet drives transcription from a site downstream of the common TSS, perhaps because it is too close to that TSS. In all, it appears that the site of TAL effector-driven transcript initiation is influenced by position of the EBE and by other promoter features in a yet poorly understood way.
We discovered that the EBEs we chose for the UXO construct contain sequences apparently under selection in rice promoters, suggesting coincidence with endogenous regulatory elements. These might include elements for gene activation in response to a particular environmental or developmental cue. Introduction of an EBE with such an element into the promoter of an executor gene could lead to TAL effector-independent cell death on that cue, with potentially disastrous consequences. We examined sequences from only six EBEs, so our findings may not be generalizable. However, TAL effectors might typically be expected to target endogenous regulatory elements in the host genome, by chance because of their localization in promoters, but also because such regulatory elements would likely be relatively immutable, and thereby confer selective advantage on any corresponding TAL effector.
Although the lines included in this study were healthy and fertile under controlled growth conditions, we encountered difficulty retrieving viable, stably transformed lines for some constructs we made. In addition to the UXO construct, in which the EBEs are exactly those found in the rice genome and contain some nucleotides that do not match the RVD at that position in the corresponding TAL effector, we also made a construct containing analogous, perfectly matched EBEs. Plants with this construct were unhealthy, and no lines survived to T2. Also, we were unable to obtain plants transformed with wildtype Xa27. The latter observation, consistent with previous findings (Wu et al., 2008), suggests that the unmodified Xa27 promoter can drive expression of the executor gene and consequent cell death when outside of its native genomic context. Similarly, even with the terminator upstream, transformation events using the XOO construct only yielded one healthy line. Thus, in addition to the possibility that EBEs added to a promoter may contain regulatory elements that could drive TAL effector-independent activation, synthetic sequences, sequences out of context, and position effects might also be problematic.
In sum, though the results presented here establish the effectiveness of EBE amendment to expand the spectrum of pathogen genotypes against which an executor gene functions, multiple alternative constructs may be necessary to obtain effective, stable transformants, and all lines should undergo testing under a variety of growth conditions and field environments before distribution or commercialization. Analysis of the representation of EBE sequences in the host promoterome might be a useful preliminary to selection of EBEs, in order to exclude those that contain sequences apparently under selection. Although beyond the scope of the work we have presented here, it might also be useful to determine whether any sequences that are over- or under-represented are found in promoters of genes of a particular functional class, in order to better predict conditions or processes in which the sequence might act as a regulatory element. Taking advantage of the degeneracy in the TAL effector-DNA binding code to generate nonnative EBE sequences may enable the rational design of a promoter recognized by a desired suite of TAL effectors without the inclusion of endogenous cis elements. Finally, making the promoter modifications in situ using engineered nucleases such as TALENs (Bogdanove & Voytas, 2011) may help to guard against position effects on expression.