CRISPR-based genome editing technology is revolutionizing prokaryotic research, but it has been rarely studied in bacterial plant pathogens. Here, we have developed a targeted genome editing method ...with no requirement of donor templates for convenient and efficient gene knockout in Xanthomonas oryzae pv. oryzae (Xoo), one of the most important bacterial pathogens on rice, by employing the heterologous CRISPR/Cas12a from Francisella novicida and NHEJ proteins from Mycobacterium tuberculosis. FnCas12a nuclease generated both small and large DNA deletions at the target sites as well as it enabled multiplex genome editing, gene cluster deletion, and plasmid curing in the Xoo PXO99A strain. Accordingly, a non-TAL effector-free polymutant strain PXO99AD25E, which lacks all 25 xop genes involved in Xoo pathogenesis, has been engineered through iterative genome editing. Whole-genome sequencing analysis indicated that FnCas12a did not have a noticeable off-target effect. In addition, we revealed that these strategies are also suitable for targeted genome editing in another bacterial plant pathogen Pseudomonas syringae pv. tomato (Pst). We believe that our bacterial genome editing method will greatly expand the CRISPR study on microorganisms and advance our understanding of the physiology and pathogenesis of Xoo.
As one of the most destructive bacterial phytopathogens,
causes substantial annual yield losses of many important crops. Deciphering the functional mechanisms of type III effectors, the crucial ...factors mediating
-plant interactions, will provide a valuable basis for protecting crop plants from
. Recently, the NEL (novel E3 ligase) effector RipAW was found to induce cell death on
in a E3 ligase activity-dependent manner. Here, we further deciphered the role of the E3 ligase activity in RipAW-triggered plant immunity. We found that RipAW
, the E3 ligase mutant of RipAW, could not induce cell death but retained the ability of triggering plant immunity in
, indicating that the E3 ligase activity is not essential for RipAW-triggered immunity. By generating truncated mutants of RipAW, we further showed that the N-terminus, NEL domain and C-terminus are all required but not sufficient for RipAW-induced cell death. Furthermore, all truncated mutants of RipAW triggered ETI immune responses in
, confirming that the E3 ligase activity is not essential for RipAW-triggered plant immunity. Finally, we demonstrated that RipAW- and RipAW
-triggered immunity in
requires SGT1 (suppressor of G2 allele of
), but not EDS1 (enhanced disease susceptibility), NRG1 (N requirement gene 1), NRC (NLR required for cell death) proteins or SA (salicylic acid) pathway. Our findings provide a typical case in which the effector-induced cell death can be uncoupled with immune responses, shedding new light on effector-triggered plant immunity. Our data also provide clues for further in-depth study of mechanism underlying RipAW-induced plant immunity.
pv.
, 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
.
pv.
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
pv.
strain PXO99
lacking
,
, and
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.
The soil-borne fungal pathogen Verticillium dahliae Kleb causes Verticillium wilt in a wide range of crops including cotton (Gossypium hirsutum). To date, most upland cotton varieties are susceptible ...to V. dahliae and the breeding for cotton varieties with the resistance to Verticillium wilt has not been successful.
Hpa1Xoo is a harpin protein from Xanthomonas oryzae pv. oryzae which induces the hypersensitive cell death in plants. When hpa1Xoo was transformed into the susceptible cotton line Z35 through Agrobacterium-mediated transformation, the transgenic cotton line (T-34) with an improved resistance to Verticillium dahliae was obtained. Cells of the transgenic T-34, when mixed with the conidia suspension of V. dahliae, had a higher tolerance to V. dahliae compared to cells of untransformed Z35. Cells of T-34 were more viable 12 h after mixing with V. dahliae conidia suspension. Immunocytological analysis showed that Hpa1Xoo, expressed in T-34, accumulated as clustered particles along the cell walls of T-34. In response to the infection caused by V. dahliae, the microscopic cell death and the generation of reactive oxygen intermediates were observed in leaves of T-34 and these responses were absent in leaves of Z35 inoculated with V. dahliae. Quantitative RT-PCR analysis indicated that five defense-related genes, ghAOX1, hin1, npr1, ghdhg-OMT, and hsr203J, were up-regulated in T-34 inoculated with V. dahliae. The up-regulations of these defense-relate genes were not observed or in a less extent in leaves of Z-35 after the inoculation.
Hpa1Xoo accumulates along the cell walls of the transgenic T-34, where it triggers the generation of H2O2 as an endogenous elicitor. T-34 is thus in a primed state, ready to protect the host from the pathogen. The results of this study suggest that the transformation of cotton with hpa1Xoo could be an effective approach for the development of cotton varieties with the improved resistance against soil-borne pathogens.
Abstract
Transcription activator-like effectors (TALEs) produced by plant pathogenic bacteria mainly belonging to the genus
Xanthomonas
cause plant diseases through activation of host susceptibility ...genes in plant cell nuclei. How TALEs enter plant cell nuclei was not clear until recent studies about PthXo1 and TALI, two TALEs produced by
Xanthomonas oryzae
pv.
oryzae
(
Xoo
) and
X. oryzae
pv.
oryzicola
(
Xoc
), the rice (
Oryza sativa
) pathogens that cause bacterial blight and bacterial leaf streak, respectively. Here, we report that rice importin IMPα1b serves as a nuclear transport receptor in rice plants to facilitate the nuclear import of PthXo1 and TALI from
Xoo
and
Xoc
, respectively. While wild-type (WT) rice plants support the nuclear import of PthXo1 and TALI, nuclear trafficking is defective in
OsIMPα1b
loss-of-function mutants generated by clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated 9 (Cas9)-based gene editing. In the edited plants infected by
Xoo
, OsIMPα1b sequesters PthXo1 from the nucleus, the PthXo1-targeted rice susceptibility gene is no longer activated, and bacterial virulence and blight disease are alleviated as a result. In the edited plants infected by
Xoc
, OsIMPα1b sequesters TALI from the nucleus, the role of TALI in suppressing rice defense responses is nullified, and rice defense responses are in turn activated to inhibit bacterial virulence and alleviate bacterial leaf streak severity.
The transcriptome profile in leaves and roots of the transgenic cotton line T-34 expressing hpa1Xoo from Xanthomonas oryzae pv. oryzae was analysed using a customized 12k cotton cDNA microarray. A ...total of 530 cDNA transcripts involved in 34 pathways were differentially expressed in the transgenic line T-34, in which 123 differentially expressed genes were related to the cotton defence responses including the hypersensitive reaction, defence responses associated with the recognition of pathogen-derived elicitors, and defence signalling pathways mediated by salicylic acid, jasmonic acid, ethylene, auxin, abscicic acid, and Ca²⁺. Furthermore, transcripts encoding various leucine-rich protein kinases and mitogen-activated protein kinases were up-regulated in the transgenic line T-34 and expression of transcripts related to the energy producing and consuming pathway was also increased, which suggested that the enhanced metabolism related to the host defence response in the transgenic line T-34 imposed an increased energy demand on the transgenic plant.
Xanthomonas oryzae pv. oryzae depends on a type III secretion system (T3SS) to translocate effectors into host cells for its ability to cause bacterial blight of rice. All type III (T3) effectors ...with known function in X. oryzae pv. oryzae belong to a family of transcription activator-like (TAL) effectors. However, other, non-TAL-related effector genes are present in the genome, although their role in virulence and their mode of action have yet to be elucidated. Here, we report the generation of mutants for 18 non-TAL T3 effector genes and the identification of one that contributes to the virulence of strain PXO99(A). XopZ(PXO99) encodes a predicted 1,414-amino-acid protein of unknown function. PXO99(A) contains two identical copies of the gene due to a duplication of 212 kb in the genome. Strains with knockout mutations of one copy of XopZ(PXO99) did not exhibit any visible virulence defect. However, strains with mutations in both copies of XopZ(PXO99) displayed reduced virulence in terms of lesion length and bacterial multiplication compared with PXO99(A). The introduction of one genomic copy of XopZ(PXO99) restores the mutant to full virulence. Transient expression of XopZ(PXO99) in Nicotiana benthamiana leaves suppresses host basal defense, which is otherwise induced by a T3SS mutant of PXO99(A), suggesting a role for XopZ(PXO99) in interfering with host innate immunity during X. oryzae pv. oryzae infection. XopZ(PXO99)-related genes are found in all Xanthomonas spp. whose genomic sequences have been determined, suggesting a conserved role for this type of effector gene in pathogenesis of Xanthomonas spp. Our results indicate that XopZ(PXO99) encodes a novel T3 effector and contributes virulence to X. oryzae pv. oryzae strains for bacterial blight of rice.
Hpa1Xoo (harpin) is a type III secreted protein of the rice blight bacterial pathogen Xanthomonas oryzae pv. oryzae that elicits a hypersensitive response (HR) in nonhost tobacco. Hpa1Xoo is ...predicted to contain two potential coiled-coil (CC) regions, one at the N-terminus with a high probability of formation, and one at the C-terminus with a lower probability of formation. We constructed several CC-equivalent peptides by a chemosynthetic method, and investigated the structure-function of the predicted Hpa1Xoo CC regions, using biophysical and biochemical approaches. Both peptides elicited an HR in tobacco. Mutant versions of the N- and C-terminal peptides that were predicted to disrupt or favor CC formation were generated. The resulting altered HR activity and oligomerization indicated that the N-terminal CC region is essential for eliciting HR, but the C-terminus is not. The results also indicate that a 14-residue fragment (LDQLLCQLISALLQ) within the N-terminal CC region is a minimal and independent functional element for HR-induction in tobacco leaves. We propose that HR-induction requires a specific oligomerization of the CC regions of Hpa1Xoo.
Xanthomonas oryzae pv. oryzae depends on a type III secretion system (T3SS) to translocate effectors into host cells for its ability to cause bacterial blight of rice. All type III (T3) effectors ...with known function in X. oryzae pv. oryzae belong to a family of transcription activator-like (TAL) effectors. However, other, non–TAL-related effector genes are present in the genome, although their role in virulence and their mode of action have yet to be elucidated. Here, we report the generation of mutants for 18 non-TAL T3 effector genes and the identification of one that contributes to the virulence of strain PXO99(A). XopZ(PXO99) encodes a predicted 1,414-amino-acid protein of unknown function. PXO99A contains two identical copies of the gene due to a duplication of 212 kb in the genome. Strains with knockout mutations of one copy of XopZ(PXO99) did not exhibit any visible virulence defect. However, strains with mutations in both copies of XopZ(PXO99) displayed reduced virulence in terms of lesion length and bacterial multiplication compared with PXO99(A). The introduction of one genomic copy of XopZ(PXO99) restores the mutant to full virulence. Transient expression of XopZ(PXO99) in Nicotiana benthamiana leaves suppresses host basal defense, which is otherwise induced by a T3SS mutant of PXO99(A), suggesting a role for XopZ(PXO99) in interfering with host innate immunity during X. oryzae pv. oryzae infection. XopZ(PXO99)-related genes are found in all Xanthomonas spp. whose genomic sequences have been determined, suggesting a conserved role for this type of effector gene in pathogenesis of Xanthomonas spp. Our results indicate that XopZ(PXO99) encodes a novel T3 effector and contributes virulence to X. oryzae pv. oryzae strains for bacterial blight of rice.
Many species of plant-pathogenic gram-negative bacteria deploy the type III (T3) secretion system to secrete virulence components, which are mostly characteristic of protein effectors targeting the ...cytosol of the plant cell following secretion.
pv.
(
), a rice pathogen causing bacterial blight disease, uses the T3 accessory protein HrpE to assemble the pilus pathway, which in turn secretes transcription activator-like (TAL) effectors. The
gene can execute extensive physiological and pathological functions beyond effector secretion. As evidenced in this study, when the
gene was deleted from the
genome, the bacteria incur seriouimpairments in multiplication, motility, and virulence. The virulence nullification is attributed to reduced secretion and translocation of PthXo1, which is a TAL effector that determines the bacterial virulence in the susceptible rice varieties. When the HrpE protein produced by prokaryotic expression is applied to plants, the recombinant protein is highly effective at inducing the defense response. Moreover, leaf photosynthesis efficiency is enhanced in HrpE-treated plants. These results provide experimental avenues to modulate the plant defense and growth tradeoff by manipulating a bacterial T3 accessory protein.
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