Bacterial Leaf Streak Diseases of Plants: Symptom Convergence in Monocot Plants by Distant Pathogenic Xanthomonas Species.

Bacterial leaf streak (BLS) is a disease of monocot plants caused by Xanthomonas translucens on small grains, X. vasicola on maize and sorghum, and X. oryzae on rice. These three pathogens cause remarkably similar symptomology in their host plants. Despite causing similar symptoms, BLS pathogens are dispersed throughout the larger Xanthomonas phylogeny. Each aforementioned species includes strain groups that do not cause BLS and instead cause vascular disease. In this commentary, we hypothesize that strains of X. translucens, X. vasicola, and X. oryzae convergently evolved to cause BLS due to shared evolutionary pressures. We examined the diversity of secreted effectors, which may be important virulence factors for BLS pathogens and their evolution. We discuss evidence that differences in gene regulation and abilities to manipulate plant hormones may also separate BLS pathogens from other Xanthomonas species or pathovars. BLS is becoming an increasing issue across the three pathosystems. Overall, we hope that a better understanding of conserved mechanisms used by BLS pathogens will enable researchers to translate findings across production systems and guide approaches to control this (re)emerging threat.

TAL Effectors with Avirulence Activity in African Strains of Xanthomonas oryzae pv. oryzae.

BACKGROUND: Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight, a devastating disease of rice. Among the type-3 effectors secreted by Xoo to support pathogen virulence, the Transcription Activator-Like Effector (TALE) family plays a critical role. Some TALEs are major virulence factors that activate susceptibility (S) genes, overexpression of which contributes to disease development. Host incompatibility can result from TALE-induced expression of so-called executor (E) genes leading to a strong and rapid resistance response that blocks disease development. In that context, the TALE functions as an avirulence (Avr) factor. To date no such avirulence factors have been identified in African strains of Xoo. RESULTS: With respect to the importance of TALEs in the Rice-Xoo pathosystem, we aimed at identifying those that may act as Avr factor within African Xoo. We screened 86 rice accessions, and identified 12 that were resistant to two African strains while being susceptible to a well-studied Asian strain. In a gain of function approach based on the introduction of each of the nine tal genes of the avirulent African strain MAI1 into the virulent Asian strain PXO99A, four were found to trigger resistance on specific rice accessions. Loss-of-function mutational analysis further demonstrated the avr activity of two of them, talD and talI, on the rice varieties IR64 and CT13432 respectively. Further analysis of TalI demonstrated the requirement of its activation domain for triggering resistance in CT13432. Resistance in 9 of the 12 rice accessions that were resistant against African Xoo specifically, including CT13432, could be suppressed or largely suppressed by trans-expression of the truncTALE tal2h, similarly to resistance conferred by the Xa1 gene which recognizes TALEs generally independently of their activation domain. CONCLUSION: We identified and characterized TalD and TalI as two African Xoo TALEs with avirulence activity on IR64 and CT13432 respectively. Resistance of CT13432 against African Xoo results from the combination of two mechanisms, one relying on the TalI-mediated induction of an unknown executor gene and the other on an Xa1-like gene or allele.

Confirmation report of Bacterial Leaf Streak disease of rice caused by Xanthomonas oryzae pv. oryzicola in Senegal.

Xanthomonas oryzae pv. X. oryzicola (Xoc), the causal agent of Bacterial Leaf Streak (BLS), is considered as one of the most important emerging pathogens of rice in Africa. This disease is estimated as responsible of 20 to 30% yield loss (Sileshi et Gebeyehu 2021) and has been characterized in several west African countries including Mali and Burkina Faso since 2003 and more recently in Ivory Coast (Wonni et al. 2014, Diallo et al. 2021). Presence of BLS symptoms in Senegal were reported by Trinh in 1980 but, to our knowledge, BLS occurrence has never been validated further and no strain of Xoc have ever been isolated from Senegalese rice fields. Xoc is transmitted by seeds which contribute to its spread through the rice trade (Sileshi et Gebeyehu 2021). To confirm Trinh's observations, we surveyed rice fields between 2014 and 2016 in eight different regions where rice is produced in Senegal. Typical disease symptoms characterized by yellow-brown to black translucent leaf streaks sometimes along with exudates, were detected in fields of several regions and collected. Leaf pieces were successively sanitized, rinsed in sterile water, and symptomatic fragments were ground using the Qiagen Tissue Lyser System (QIAGEN, Courtaboeuf, France). The leaf powder was diluted in 1.5 ml of sterile water and incubated for 30 minutes at room temperature. Ten µl of the suspension was streaked on semi-selective PSA medium and incubated at 28°C for 3 to 7 days. Characteristic round, convex, mucous, straw-yellow Xoc candidate colonies were purified from six individual leaf samples from three distinct sites in the northern Senegal River Valley. To confirm their identity, isolated strains were tested for pathogenicity and molecular characterization. All isolates were subjected to the multiplex PCR developed for the identification of X. oryzae pathovars (Lang et al., 2010) and revealed the same PCR profile (two amplicons of 324 and 691 base pairs) similar to that of the Xoc reference strain BLS256. Leaves of 5-week-old plants of O. sativa cv. Kitaake were infiltrated with a needleless syringe containing a bacterial suspension set at an optical density of 0.5. Upon seven days of incubation under greenhouse conditions (27 ± 1°C with a 12-hour photoperiod), all infiltrated spots (2 spots on 3 plants per isolate) developed water-soaked lesions similar to those caused by control strain BLS256, except when leaves were infiltrated with water. Symptomatic leaf tissues were ground and plated on PSA medium, resulting in colonies with typical Xanthomonas morphology that were diagnosed as Xoc by multiplex PCR typing, thus fulfilling Koch's postulate. At last, four of the isolates were subjected to gyrB sequencing upon PCR amplification using the universal primers XgyrB1F and XgyrB1R (Young et al., 2008). Analysis of 780bp partial gyrB sequences of strains S18-3-4, S23-1-12, S52-1-4 and S52-1-10 highlighted 100% identity with the gyrB sequence of strain BLS256 (Acc. No. CP003057). To our knowledge, this is the first report of BLS in Senegal which is supported by molecular characterization methods. This study validates the presence of BLS in Senegal and will serve as a basis for future efforts of rice breeding for locally adapted resistance. More studies are needed to clarify the spatial distribution and prevalence of BLS in Senegal as rice cultivation is expanding rapidly in the country.

Cloning of the Rice Xo1 Resistance Gene and Interaction of the Xo1 Protein with the Defense-Suppressing Xanthomonas Effector Tal2h.

The Xo1 locus in the heirloom rice variety Carolina Gold Select confers resistance to bacterial leaf streak and bacterial blight, caused by Xanthomonas oryzae pv. oryzicola and X. oryzae pv. oryzae, respectively. Resistance is triggered by pathogen-delivered transcription activator-like effectors (TALEs) independent of their ability to activate transcription and is suppressed by truncated variants called truncTALEs, common among Asian strains. By transformation of the susceptible variety Nipponbare, we show that one of 14 nucleotide-binding, leucine-rich repeat (NLR) protein genes at the locus, with a zinc finger BED domain, is the Xo1 gene. Analyses of published transcriptomes revealed that the Xo1-mediated response is more similar to those mediated by two other NLR resistance genes than it is to the response associated with TALE-specific transcriptional activation of the executor resistance gene Xa23 and that a truncTALE dampens or abolishes activation of defense-associated genes by Xo1. In Nicotiana benthamiana leaves, fluorescently tagged Xo1 protein, like TALEs and truncTALEs, localized to the nucleus. And endogenous Xo1 specifically coimmunoprecipitated from rice leaves with a pathogen-delivered, epitope-tagged truncTALE. These observations suggest that suppression of Xo1-function by truncTALEs occurs through direct or indirect physical interaction. They further suggest that effector coimmunoprecipitation may be effective for identifying or characterizing other resistance genes.

Functional analysis of African Xanthomonas oryzae pv. oryzae TALomes reveals a new susceptibility gene in bacterial leaf blight of rice.

Most Xanthomonas species translocate Transcription Activator-Like (TAL) effectors into plant cells where they function like plant transcription factors via a programmable DNA-binding domain. Characterized strains of rice pathogenic X. oryzae pv. oryzae harbor 9-16 different tal effector genes, but the function of only a few of them has been decoded. Using sequencing of entire genomes, we first performed comparative analyses of the complete repertoires of TAL effectors, herein referred to as TALomes, in three Xoo strains forming an African genetic lineage different from Asian Xoo. A phylogenetic analysis of the three TALomes combined with in silico predictions of TAL effector targets showed that African Xoo TALomes are highly conserved, genetically distant from Asian ones, and closely related to TAL effectors from the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Nine clusters of TAL effectors could be identified among the three TALomes, including three showing higher levels of variation in their repeat variable diresidues (RVDs). Detailed analyses of these groups revealed recombination events as a possible source of variation among TAL effector genes. Next, to address contribution to virulence, nine TAL effector genes from the Malian Xoo strain MAI1 and four allelic variants from the Burkinabe Xoo strain BAI3, thus representing most of the TAL effector diversity in African Xoo strains, were expressed in the TAL effector-deficient X. oryzae strain X11-5A for gain-of-function assays. Inoculation of the susceptible rice variety Azucena lead to the discovery of three TAL effectors promoting virulence, including two TAL effectors previously reported to target the susceptibility (S) gene OsSWEET14 and a novel major virulence contributor, TalB. RNA profiling experiments in rice and in silico prediction of EBEs were carried out to identify candidate targets of TalB, revealing OsTFX1, a bZIP transcription factor previously identified as a bacterial blight S gene, and OsERF#123, which encodes a subgroup IXc AP2/ERF transcription factor. Use of designer TAL effectors demonstrated that induction of either gene resulted in greater susceptibility to strain X11-5A. The induction of OsERF#123 by BAI3Δ1, a talB knockout derivative of BAI3, carrying these designer TAL effectors increased virulence of BAI3Δ1, validating OsERF#123 as a new, bacterial blight S gene.

Suppression of Xo1-Mediated Disease Resistance in Rice by a Truncated, Non-DNA-Binding TAL Effector of Xanthomonas oryzae.

Delivered into plant cells by type III secretion from pathogenic Xanthomonas species, TAL (transcription activator-like) effectors are nuclear-localized, DNA-binding proteins that directly activate specific host genes. Targets include genes important for disease, genes that confer resistance, and genes inconsequential to the host-pathogen interaction. TAL effector specificity is encoded by polymorphic repeats of 33-35 amino acids that interact one-to-one with nucleotides in the recognition site. Activity depends also on N-terminal sequences important for DNA binding and C-terminal nuclear localization signals (NLS) and an acidic activation domain (AD). Coding sequences missing much of the N- and C-terminal regions due to conserved, in-frame deletions are present and annotated as pseudogenes in sequenced strains of Xanthomonas oryzae pv. oryzicola (Xoc) and pv. oryzae (Xoo), which cause bacterial leaf streak and bacterial blight of rice, respectively. Here we provide evidence that these sequences encode proteins we call "truncTALEs," for "truncated TAL effectors." We show that truncTALE Tal2h of Xoc strain BLS256, and by correlation truncTALEs in other strains, specifically suppress resistance mediated by the Xo1 locus recently described in the heirloom rice variety Carolina Gold. Xo1-mediated resistance is triggered by different TAL effectors from diverse X. oryzae strains, irrespective of their DNA binding specificity, and does not require the AD. This implies a direct protein-protein rather than protein-DNA interaction. Similarly, truncTALEs exhibit diverse predicted DNA recognition specificities. And, in vitro, Tal2h did not bind any of several potential recognition sites. Further, a single candidate NLS sequence in Tal2h was dispensable for resistance suppression. Many truncTALEs have one 28 aa repeat, a length not observed previously. Tested in an engineered TAL effector, this repeat required a single base pair deletion in the DNA, suggesting that it or a neighbor disengages. The presence of the 28 aa repeat, however, was not required for resistance suppression. TruncTALEs expand the paradigm for TAL effector-mediated effects on plants. We propose that Tal2h and other truncTALEs act as dominant negative ligands for an immune receptor encoded by the Xo1 locus, likely a nucleotide binding, leucine-rich repeat protein. Understanding truncTALE function and distribution will inform strategies for disease control.

Ectopic activation of the rice NLR heteropair RGA4/RGA5 confers resistance to bacterial blight and bacterial leaf streak diseases.

Bacterial blight (BB) and bacterial leaf streak (BLS) are important diseases in Oryza sativa caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), respectively. In both bacteria, transcription activator-like (TAL) effectors are major virulence determinants that act by transactivating host genes downstream of effector-binding elements (EBEs) bound in a sequence-specific manner. Resistance to Xoo is mostly related to the action of TAL effectors, either by polymorphisms that prevent the induction of susceptibility (S) genes or by executor (R) genes with EBEs embedded in their promoter, and that induce cell death and resistance. For Xoc, no resistance sources are known in rice. Here, we investigated whether the recognition of effectors by nucleotide binding and leucine-rich repeat domain immune receptors (NLRs), the most widespread resistance mechanism in plants, is also able to stop BB and BLS. In one instance, transgenic rice lines harboring the AVR1-CO39 effector gene from the rice blast fungus Magnaporthe oryzae, under the control of an inducible promoter, were challenged with transgenic Xoo and Xoc strains carrying a TAL effector designed to transactivate the inducible promoter. This induced AVR1-CO39 expression and triggered BB and BLS resistance when the corresponding Pi-CO39 resistance locus was present. In a second example, the transactivation of an auto-active NLR by Xoo-delivered designer TAL effectors resulted in BB resistance, demonstrating that NLR-triggered immune responses efficiently control Xoo. This forms the foundation for future BB and BLS disease control strategies, whereupon endogenous TAL effectors will target synthetic promoter regions of Avr or NLR executor genes.

A knowledge-based molecular screen uncovers a broad-spectrum OsSWEET14 resistance allele to bacterial blight from wild rice.

Transcription activator-like (TAL) effectors are type III-delivered transcription factors that enhance the virulence of plant pathogenic Xanthomonas species through the activation of host susceptibility (S) genes. TAL effectors recognize their DNA target(s) via a partially degenerate code, whereby modular repeats in the TAL effector bind to nucleotide sequences in the host promoter. Although this knowledge has greatly facilitated our power to identify new S genes, it can also be easily used to screen plant genomes for variations in TAL effector target sequences and to predict for loss-of-function gene candidates in silico. In a proof-of-principle experiment, we screened a germplasm of 169 rice accessions for polymorphism in the promoter of the major bacterial blight susceptibility S gene OsSWEET14, which encodes a sugar transporter targeted by numerous strains of Xanthomonas oryzae pv. oryzae. We identified a single allele with a deletion of 18 bp overlapping with the binding sites targeted by several TAL effectors known to activate the gene. We show that this allele, which we call xa41(t), confers resistance against half of the tested Xoo strains, representative of various geographic origins and genetic lineages, highlighting the selective pressure on the pathogen to accommodate OsSWEET14 polymorphism, and reciprocally the apparent limited possibilities for the host to create variability at this particular S gene. Analysis of xa41(t) conservation across the Oryza genus enabled us to hypothesize scenarios as to its evolutionary history, prior to and during domestication. Our findings demonstrate that resistance through TAL effector-dependent loss of S-gene expression can be greatly fostered upon knowledge-based molecular screening of a large collection of host plants.

Corrigendum: MorTALKombat: the story of defense against TAL effectors through loss-of-susceptibility.

[This corrects the article on p. 535 in vol. 6, PMID: 26236326.].

MorTAL Kombat: the story of defense against TAL effectors through loss-of-susceptibility.

Many plant-pathogenic xanthomonads rely on Transcription Activator-Like (TAL) effectors to colonize their host. This particular family of type III effectors functions as specific plant transcription factors via a programmable DNA-binding domain. Upon binding to the promoters of plant disease susceptibility genes in a sequence-specific manner, the expression of these host genes is induced. However, plants have evolved specific strategies to counter the action of TAL effectors and confer resistance. One mechanism is to avoid the binding of TAL effectors by mutations of their DNA binding sites, resulting in resistance by loss-of-susceptibility. This article reviews our current knowledge of the susceptibility hubs targeted by Xanthomonas TAL effectors, possible evolutionary scenarios for plants to combat the pathogen with loss-of-function alleles, and how this knowledge can be used overall to develop new pathogen-informed breeding strategies and improve crop resistance.

Five phylogenetically close rice SWEET genes confer TAL effector-mediated susceptibility to Xanthomonas oryzae pv. oryzae.

Bacterial plant-pathogenic Xanthomonas strains translocate transcription activator-like (TAL) effectors into plant cells to function as specific transcription factors. Only a few plant target genes of TAL effectors have been identified, so far. Three plant SWEET genes encoding putative sugar transporters are known to be induced by TAL effectors from rice-pathogenic Xanthomonas oryzae pv. oryzae (Xoo). We predict and validate that expression of OsSWEET14 is induced by a novel TAL effector, Tal5, from an African Xoo strain. Artificial TAL effectors (ArtTALs) were constructed to individually target 20 SWEET orthologs in rice. They were used as designer virulence factors to study which rice SWEET genes can support Xoo virulence. The Tal5 target box differs from those of the already known TAL effectors TalC, AvrXa7 and PthXo3, which also induce expression of OsSWEET14, suggesting evolutionary convergence on key targets. ArtTALs efficiently complemented an Xoo talC mutant, demonstrating that specific induction of OsSWEET14 is the key target of TalC. ArtTALs that specifically target individual members of the rice SWEET family revealed three known and two novel SWEET genes to support bacterial virulence. Our results demonstrate that five phylogenetically close SWEET proteins, which presumably act as sucrose transporters, can support Xoo virulence.