Rice aquaporin PIP1;3 and harpin Hpa1 of bacterial blight pathogen cooperate in a type III effector translocation.

Varieties of Gram-negative bacterial pathogens infect their eukaryotic hosts by deploying the type III translocon to deliver effector proteins into the cytosol of eukaryotic cells in which effectors execute their pathological functions. The translocon is hypothetically assembled by bacterial translocators in association with the assumed receptors situated on eukaryotic plasma membranes. This hypothesis is partially verified in the present study with genetic, biochemical, and pathological evidence for the role of a rice aquaporin, plasma membrane intrinsic protein PIP1;3, in the cytosolic import of the transcription activator-like effector PthXo1 from the bacterial blight pathogen. PIP1;3 interacts with the bacterial translocator Hpa1 at rice plasma membranes to control PthXo1 translocation from cells of a well-characterized strain of the bacterial blight pathogen into the cytosol of cells of a susceptible rice variety. An extracellular loop sequence of PIP1;3 and the α-helix motif of Hpa1 determine both the molecular interaction and its consequences with respect to the effector translocation and the bacterial virulence on the susceptible rice variety. Overall, these results provide multiple experimental avenues to support the hypothesis that interactions between bacterial translocators and their interactors at the target membrane are essential for bacterial effector translocation.

Gene targeting by the TAL effector PthXo2 reveals cryptic resistance gene for bacterial blight of rice.

Bacterial blight of rice is caused by the γ-proteobacterium Xanthomonas oryzae pv. oryzae, which utilizes a group of type III TAL (transcription activator-like) effectors to induce host gene expression and condition host susceptibility. Five SWEET genes are functionally redundant to support bacterial disease, but only two were experimentally proven targets of natural TAL effectors. Here, we report the identification of the sucrose transporter gene OsSWEET13 as the disease-susceptibility gene for PthXo2 and the existence of cryptic recessive resistance to PthXo2-dependent X. oryzae pv. oryzae due to promoter variations of OsSWEET13 in japonica rice. PthXo2-containing strains induce OsSWEET13 in indica rice IR24 due to the presence of an unpredicted and undescribed effector binding site not present in the alleles in japonica rice Nipponbare and Kitaake. The specificity of effector-associated gene induction and disease susceptibility is attributable to a single nucleotide polymorphism (SNP), which is also found in a polymorphic allele of OsSWEET13 known as the recessive resistance gene xa25 from the rice cultivar Minghui 63. The mutation of OsSWEET13 with CRISPR/Cas9 technology further corroborates the requirement of OsSWEET13 expression for the state of PthXo2-dependent disease susceptibility to X. oryzae pv. oryzae. Gene profiling of a collection of 104 strains revealed OsSWEET13 induction by 42 isolates of X. oryzae pv. oryzae. Heterologous expression of OsSWEET13 in Nicotiana benthamiana leaf cells elevates sucrose concentrations in the apoplasm. The results corroborate a model whereby X. oryzae pv. oryzae enhances the release of sucrose from host cells in order to exploit the host resources.

Tobacco TTG2 regulates vegetative growth and seed production via the predominant role of ARF8 in cooperation with ARF17 and ARF19.

BACKGROUND: Plant TRANSPARENT TESTA GLABRA (TTG) proteins regulate various developmental activities via the auxin signaling pathway. Recently, we elucidated the developmental role of tobacco (Nicotiana tabacum L.) NtTTG2 in association with 12 genes that putatively encode AUXIN RESPONSIVE FACTOR (ARF) proteins, including NtARF8, NtARF17, and NtARF19. Here we show that NtTTG2 regulates tobacco growth and development by involving the NtARF8, NtARF17, and NtARF19 genes, with the NtARF8 gene playing a predominant contribution. RESULTS: Independent silencing of the NtARF8 gene more strongly repressed tobacco growth than silencing the NtARF17 or NtARF19 gene and more effectively eradicated the growth enhancement effect of NtTTG2 overexpression. In contrast, plant growth was not affected by silencing additional nine NtTTG2-regulated NtARF genes. In double and triple gene silencing combinations, silencing the NtARF8 gene was more effective than silencing the NtARF17 or NtARF19 gene to repress growth as well as nullify growth enhancement. Therefore, the NtARF8 predominantly cooperated with the NtARF17 and NtAFR19 of the NtTTG2 functional pathway. NtARF8 also contributed to NtTTG2-regulated seed production as concurrent NtTTG2 and NtARF8 overexpression played a synergistic role in seed production quantity, whereas concurrent silencing of both genes caused more severe seed abortion than single gene silencing. In plant cells, the NtTTG2 protein facilitated the nuclear import of NtARF8 as well as increased its function as a transcription activator. CONCLUSIONS: NtARF8 is an integral component of the NtTTG2 functional pathway, which regulates tobacco growth and development.

A type VI secretion system effector TseG of Pantoea ananatis is involved in virulence and antibacterial activity.

The type VI secretion system (T6SS) of many gram-negative bacteria injects toxic effectors into adjacent cells to manipulate host cells during pathogenesis or to kill competing bacteria. However, the identification and function of the T6SS effectors remains only partly known. Pantoea ananatis, a gram-negative bacterium, is commonly found in various plants and natural environments, including water and soil. In the current study, genomic analysis of P. ananatis DZ-12 causing brown stalk rot on maize demonstrated that it carries three T6SS gene clusters, namely, T6SS-1, T6SS-2, and T6SS-3. Interestingly, only T6SS-1 secretion systems are involved in pathogenicity and bacterial competition. The study also investigated the T6SS-1 system in detail and identified an unknown T6SS-1-secreted effector TseG by using the upstream T6SS effector chaperone TecG containing a conserved domain of DUF2169. TseG can directly interact with the chaperone TecG for delivery and with a downstream immunity protein TsiG for protection from its toxicity. TseG, highly conserved in the Pantoea genus, is involved in virulence in maize, potato, and onion. Additionally, P. ananatis uses TseG to target Escherichia coli, gaining a competitive advantage. This study provides the first report on the T6SS-1-secreted effector from P. ananatis, thereby enriching our understanding of the various types and functions of type VI effector proteins.

Functional Identification of the Xanthomonas oryzae pv. oryzae Type I-C CRISPR-Cas System and Its Potential in Gene Editing Application.

The type I clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is one of five adaptive immune systems and exists widely in bacteria and archaea. In this study, we showed that Xanthomonas oryzae pv. oryzae (Xoo) possesses a functional CRISPR system by engineering constructs mimicking its CRISPR cassette. CRISPR array analysis showed that the TTC at the 5'-end of the target sequence is a functional protospacer-adjacent motif (PAM) of CRISPR. Guide RNA (gRNA) deletion analysis identified a minimum of 27-bp spacer that was required to ensure successful self-target killing in PXO99A strain. Mutants with deletion of individual Cas genes were constructed to analyze the effects of Cas proteins on mature CRISPR RNA (crRNA), processing intermediates and DNA interference. Results showed that depleting each of the three genes, cas5d, csd1, and csd2 inactivated the pre-crRNA processing, whereas inactivation of cas3 impaired in processing pre-crRNA. Furthermore, the Xoo CRISPR/Cas system was functional in Pseudomonas syringae pv. tomato. Collectively, our results would contribute to the functional study of CRISPR/Cas system of Xoo, and also provide a new vision on the use of bacterial endogenous systems as a convenient tool for gene editing.

Identification of specific fragments of HpaG Xooc, a harpin from Xanthomonas oryzae pv. oryzicola, that induce disease resistance and enhance growth in plants.

Harpin proteins from gram-negative plant-pathogenic bacteria can stimulate hypersensitive cell death (HCD) and pathogen defense as well as enhance growth in plants. Two of these diverse activities clearly are beneficial and may depend on particular functional regions of the proteins. Identification of beneficial and deleterious regions might facilitate the beneficial use of harpin-related proteins on crops without causing negative effects like cell death. Here, we report the identification and testing of nine functional fragments of HpaG(Xooc), a 137-amino-acid harpin protein from Xanthomonas oryzae pv. oryzicola, the pathogen that causes bacterial leaf streak of rice. Polymerase chain reaction-based mutagenesis generated nine proteinaceous fragments of HpaG(Xooc); these caused different responses following their application to Nicotiana tabacum (tobacco) and Oryza sativa (rice). Fragment HpaG62-137, which spans the indicated amino acid residues of the HpaG, induced more intense HCD; in contrast, HpaG10-42 did not cause evident cell death in tobacco. However, both fragments stimulated stronger defense responses and enhanced more growth in rice than the full-length parent protein, HpaG(Xooc). Of the nine fragments, the parent protein and one deletion mutant of HpaG(Xooc) tested, HpaG10-42, stimulated higher levels of rice growth and resulted in greater levels of resistance to X. oryzae pv. oryzae and Magnaporthe grisea. These pathogens cause bacterial leaf blight and rice blast, respectively, the two most important diseases of rice world-wide. HpaG10-42 was more active than HpaG(Xooc) in inducing expression of several genes that regulate rice defense and growth processes and activating certain signaling pathways, which may explain the greater beneficial effects observed from treatment with that fragment. Overall, our results suggest that HpaG10-42 holds promise for practical agricultural use to induce disease resistance and enhance growth of rice.

A fragment of the Xanthomonas oryzae pv. oryzicola harpin HpaG Xooc reduces disease and increases yield of rice in extensive grower plantings.

Harpins of phytopathogenic bacteria stimulate defense and plant growth in many types of plants, conferring disease resistance and enhanced yield. In a previous study, we characterized nine fragments of the harpin protein HpaG(Xooc) from Xanthomonas oryzae pv. oryzicola for plant defense elicitation and plant growth stimulation activity relative to the intact protein. In plants grown under controlled conditions, the fragment HpaG10-42 was more active in both regards than HpaG(Xooc). Here, we demonstrate that the activity of HpaG10-42 in rice under field conditions significantly exceeds that of HpaG(Xooc), stimulating resistance to three important diseases and increasing grain yield. We carried out tests in 672 experimental plots with nine cultivars of rice planted at three locations. Application protocols were optimized by testing variations in application rate, frequency, and timing with respect to rice growth stage. Of the concentrations (24, 24, 12, and 6 microg/ml), and number and timing of applications (at one to four different stages of growth) tested, HpaG10-42 at 6 microg/ml applied to plants once at nursery seedling stage and three times in the field was most effective. Bacterial blight, rice blast, and sheath blight were reduced 61.6 and 56.4, 93.6 and 76.0, and 93.2 and 55.0% in indica and japonica cultivars, respectively, relative to controls. Grain yields were 22 to 27% greater. These results are similar to results obtained with typical local management practices, including use of chemicals, to decrease disease severities and increase yield in rice. Our results demonstrate that the HpaG10-42 protein fragment can be used effectively to control diseases and increase yield of this staple food crop.

Non-TAL Effectors From Xanthomonas oryzae pv. oryzae Suppress Peptidoglycan-Triggered MAPK Activation in Rice.

Xanthomonas oryzae pv. oryzae, the causal pathogen of bacterial blight of rice, depends on its type III secretion system and associated effector proteins to grow and colonize the vascular tissues of rice plants. The type III effectors include a family of closely related transcription activator-like (TAL) effectors and the rest of diverse effectors, so-called non-TAL effectors. Our understanding of non-TAL effectors for pathogenesis in rice blight is still limited. Here we report a feasible method to rapidly detect the activation of mitogen-activated protein kinase pathway in rice mesophyll protoplasts by the X. oryzae pv. oryzae derived peptidoglycan and screen for virulent effectors that can suppress the pathogen-associated molecular pattern triggered immunity (PTI) response. Amongst 17 non-TAL effectors transiently expressed in rice cells, we found that three effectors (XopZ, XopN, and XopV) were able to suppress the peptidoglycan-triggered MAPK activation. The triple mutant of the X. oryzae pv. oryzae strain PXO99A lacking XopZ, XopN, and XopV showed additively reduced virulence. Adding back either of genes restored the virulence of the triple mutant. Our results demonstrate the collective and redundant ability of defense suppression by non-TAL effectors in causing bacterial blight of rice.

Mutagenesis of PhaR, a Regulator Gene of Polyhydroxyalkanoate Biosynthesis of Xanthomonas oryzae pv. oryzae Caused Pleiotropic Phenotype Changes.

Polyhydroxyalkanoates (PHAs) are intracellular carbon and energy storage materials produced in various microorganisms under nutrient-limited conditions. PhaR is a regulatory protein involved in PHA synthesis. Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important bacterial pathogens in rice and has PHA biosynthesis genes in its genome, but the biological function of phaR in Xoo is unknown. In this study, we investigated the effects of the mutagenesis of phaR gene in Xoo strain PXO99A. Compared to the wildtype, the PhaR gene knock-out mutant PXO99ΔphaR was hypermotile and showed decreased growth rates in both rich and limited nutrient media. PXO99ΔphaR also showed almost 75% decrease in extracellular polysaccharide (EPS) production. When inoculated in rice leaves by leaf-clipping method, PXO99ΔphaR displayed reduced virulence in terms of lesion length and bacterial multiplication compared with the wildtype strain. PXO99ΔphaR also showed enhanced hypersensitive response (HR) induction in the leaves of non-host Nicotiana benthamiana with elevated hpa1 gene expression. Introduction of a cosmid containing the phaR coding sequence restored the phenotypes of the mutant to those of the wildtype strain. These results suggest that PhaR gene is an important gene that affects multiple bacterial characteristics, including EPS production, growth rate, defense response induced harpin production and motility, related to its virulence in plant.

Productivity and biochemical properties of green tea in response to full-length and functional fragments of HpaG Xooc, a harpin protein from the bacterial rice leaf streak pathogen Xanthomonas oryzae pv. oryzicola.

Harpin proteins from plant pathogenic bacteria can stimulate hypersensitive cell death (HCD), drought tolerance, defence responses against pathogens and insects in plants, as well as enhance plant growth. Recently, we identified nine functional fragments of HpaG;Xooc, a harpin protein from Xanthomonas oryzae pv.oryzicola, the pathogen that causes bacterial leaf streak in rice. Fragments HpaG;1-94'HpaG;10-42, and HpaG;62-138, which contain the HpaG;Xooc regions of the amino acid sequence as indicated by the number spans, exceed the parent protein in promoting growth, pathogen defence and HCD in plants. Here we report improved productivity and biochemical properties of green tea (Camellia sinensis) in response to the fragments tested in comparison with HpaG;Xooc and an inactive protein control. Field tests suggested that the four proteins markedly increased the growth and yield of green tea, and increased the leaf content of tea catechols, a group of compounds that have relevance in the prevention and treatment of human diseases. In particular, HpaG;1-94 was more active than HpaG;Xooc in expediting the growth of juvenile buds and leaves used as green tea material and increased the catechol content of processed teas. When tea shrubs were treated with HpaH;Xooc and HpaG;1-94 compared with a control, green tea yields were over 55% and 39% greater, and leaf catechols were increased by more than 64% and 72%, respectively. The expression of three homologues of the expansin genes, which regulate plant cell growth, and the CsCHS gene encoding a tea chalcone synthase, which critically regulates the biosynthesis of catechols, were induced in germinal leaves of tea plants following treatment with HpaG;1-94 or HpaG;Xooc. Higher levels of gene expression were induced by the application of HpaG;1-94 than HpaG;Xooc. Our results suggest that the harpin protein, especially the functional fragment HpaG;1-94, can be used to effectively increase the yield and improve the biochemical properties of green tea, a drink with medicinal properties.

Combinative effects of a bacterial type-III effector and a biocontrol bacterium on rice growth and disease resistance.

Expression of HpaG(Xoo), a bacterial type-III effector, in transgenic plants induces disease resistance. Resistance also can be elicited by biocontrol bacteria. In both cases, plant growth is often promoted. Here we address whether biocontrol bacteria and HpaG(Xoo) can act together to provide better results in crop improvement. We studied effects of Pseudomonas cepacia on the rice variety R109 and the hpaG(Xoo)-expressing rice line HER1. Compared to R109, HER1 showed increased growth, grain yield, and defense responses toward diseases and salinity stress. Colonization of roots by P. cepacia caused 20% and 13% increase, in contrast to controls, in root growth of R109 and HER1. Growth of leaves and stems also increased in R109 but that of HER1 was inhibited. When P. cepacia colonization was subsequent to plant inoculation with Rhizoctonia solani, a pathogen that causes sheath blight, the disease was less severe than controls in both R109 and HER1; HER1, nevertheless, was more resistant, suggesting that P. cepacia and HpaG(Xoo) cooperate in inducing disease resistance. Several genes that critically regulate growth and defense behaved differentially in HER1 and R109 while responding to P. cepacia. In R109 leaves, the OsARF1 gene, which regulates plant growth, was expressed in consistence with growth promotion by P. cepacia. Inversely, OsARF1 expression was coincident with inhibition in growth of HER1 leaves. In both plants, the expression of OsEXP1, which encodes an expansin protein involved in plant growth,was concomitant with growth promotion in leaves instead of roots,in response to P. cepacia . We also studied OsMAPK, a gene that encodes a mitogen-activated protein kinase and controls defense responses toward salinity and infection by pathogens in rice. In response to P. cepacia, an early expression of OsMAPK was coincident with R109 resistance to the disease, while HER1 expressed the gene similarly whether P. cepacia was present or not. Evidently, P. cepacia and G(Xoo)-gene mediated resistance may act differently in rice growth and resistance. Whereas combinative effects of P. cepacia and HpaG(Xoo) in disease resistance have a great potential in agricultural use, it is interesting to study mechanisms that underlie interactions involving biocontrol bacteria, type-III effectors and pathogens.

Xanthomonas oryzae pv. oryzae requires H-NS-family protein XrvC to regulate virulence during rice infection.

Histone-like nucleoid-structuring (H-NS) proteins, which are conserved in Gram-negative bacteria, bind DNA and act as the global transcriptional repressors. In this study, we identified and characterized the xrvC gene encoding a H-NS protein in Xathomonas oryzae pv. oryzae (Xoo) Philippines strain PXO99(A) Compared with the wild type, the xrvC-deficient mutant of PXO99(A) (named PXO99ΔxrvC) showed a reduced growth rate in both nutrient-rich and nutrient-limited media. Interestingly, PXO99ΔxrvC exhibited significantly reduced virulence on rice cultivar IRBB214, but its virulence on 31 other rice cultivars was not affected. Transcriptional analysis revealed that the expression of hrpG, hrpX and hpa1 and of 15 out of 18 tested non-TAL (transcription activator-like) effector genes was decreased significantly in the xrvC mutant compared with that in the wild type. In addition, loss of xrvC also impaired the induction of the rice susceptibility gene Os8N3 in IRBB214 by PXO99(A) Our results suggest that the xrvC gene is involved in bacterial growth, and it plays a vital role in virulence by positively regulating the expression of hrp genes and non-TAL effector genes in PXO99(A) and the susceptibility gene Os8N3 in rice.

Genetic diversity of harpins from Xanthomonas oryzae and their activity to induce hypersensitive response and disease resistance in tobacco.

Three hrfA (hypersensitive response-functioning faction A) homologues (hrfl, hrf2 and hrf3) are cloned from 12 strains of Xanthomonas oryzae using PCR based techniques. Hrf1, hrf2 and hrf3 are derived from strains belonging to X. o. pv. oryzae, X. o. pv. oryzicola and X. o. pv. oryzae respectively. Sequence analysis shows that all three genes encode glycine-rich proteins with various numbers of GGG-GG motifs. They all share a conserved cysteine residue at position 45 or 47. Hrf1 and hrf3 encode Harpin(xoo) while hrf2 encodes Harpin(xooc) Hrf1 and hrf3 encodes two different types of Harpin(xoo) proteins. Hrf1 from X. o. pv. oryzae strains (JxoIII, JxoIV, Jxov, Pxo61, Pxo76, Pxo79, Pxo99, Pxo99 and Pxo124) encodes a 15.6 kD Harpin(xoo) with 3 GGG-GG motifs while Hrf3 from strain Pxo86 and Pxo112 encodes a 15.9 kD Harpin(xoo) with 4 GGG-GG motifs. Harpin(xooc) encoded by hrf2 from X. o. pv. oryzicola (strain RS105) has the molecular weight of 15.3 kD and contains 2 GGG-GG motifs. Cluster analysis is performed using deduced sequences of hrf1, hrf2 and hrf3 as well as previously reported Hpa1 and Xopl protein sequence. The results indicated that Harpin(xoo) and Harpin(xooc) belong to two closely related subgroups. Hrf, hrf2 and hrf3 are expressed in E. coli strain BL21 successfully. Under the same condition, hrf1, hrf2 and hrf3 are expressed at the level of 0.389, 0.530 and 0.083 mg/mL respectively. All expressed hrf1, hrf2 and hrf3 proteins (Harpins) are shown to be able to induce hypersensitive reaction and TMV resistance on tobacco. Among the three proteins, Hrf2 has the highest activity while Hrf3 has the lowest activity.