Bacillus cereus AR156 Extracellular Polysaccharides Served as a Novel Micro-associated Molecular Pattern to Induced Systemic Immunity to Pst DC3000 in Arabidopsis.
ABSTRACT: Non-host resistance (NHR) is a broad-spectrum plant defense. Upon colonizing on the surface on the root or leaves of non-host species, pathogens initial encounter preform and induce defense response in plant, such as induced hypersensitive response, PAMPs triggered immunity (PTI), and effector triggered immunity (ETI). The ability of plants to develop an induced systemic response (ISR) in reaction to the colonization by non-pathogenic rhizobacterium depends on interactions between host plants and the colonizing rhizobacterium, and the ISR also can be defined as a NHR. However, how the colonization signal is and how systemic resistance to pathogens is developed is still unclear. In this study, we demonstrated that the extracellular polysaccharides (EPSs) of Bacillus cereus AR156 could act as novel microbe-associated molecular patterns (MAMPs) and function in the early perception status of the ISR of B. cereus AR156. The results revealed that B. cereus AR156 EPS could induce systemic resistance to Pst DC3000 in Arabidopsis. Cellular defense response markers such as hydrogen peroxide accumulation, callose deposition, and defense-associated enzyme were induced upon challenge inoculation in the leaves primed by EPS. Moreover, the defense-related genes PR1, PR2, and PR5 and mitogen-activated kinases (MAPK) cascade marker gene MPK6 were concurrently expressed in the leaves of EPS-treated plants and induced higher resistance to Pst DC3000 in Col-0 than that in the jar1 or etr1 mutants. The protection was absent in the NahG transgenic plants and npr1 mutant, suggesting an activation of the salicylic acid (SA)- and the MAPK-dependent signaling pathways with NPR1-dependent by B. cereus AR156 EPS. In conclusion, B. cereus AR156 EPS play an important role in MAMP perception during the process of rhizobacteria-triggered NHR. This study is the first to illustrate how AR156 induces systemic resistance to Pst DC3000 in Arabidopsis. It also provides the first explanation of how plants perceive colonization of non-pathogenic bacteria and how rhizobacteria trigger ISR to plant pathogens.
Project description:Small RNAs play an important role in plant immune responses. However, their regulatory function in induced systemic resistance (ISR) is nascent. Bacillus cereus AR156 is a plant growth-promoting rhizobacterium that induces ISR in Arabidopsis against bacterial infection. Here, by comparing small RNA profiles of Pseudomonas syringae pv. tomato (Pst) DC3000-infected Arabidopsis with and without AR156 pretreatment, we identified a group of Arabidopsis microRNAs (miRNAs) that are differentially regulated by AR156 pretreatment. miR825 and miR825* are two miRNA generated from a single miRNA gene. Northern blot analysis indicated that they were significantly downregulated in Pst DC3000-infected plants pretreated with AR156, in contrast to the plants without AR156 pretreatment. miR825 targets two ubiquitin-protein ligases, while miR825* targets toll-interleukin-like receptor (TIR)-nucleotide binding site (NBS) and leucine-rich repeat (LRR) type resistance (R) genes. The expression of these target genes negatively correlated with the expression of miR825 and miR825*. Moreover, transgenic plants showing reduced expression of miR825 and miR825* displayed enhanced resistance to Pst DC3000 infection, whereas transgenic plants overexpressing miR825 and miR825* were more susceptible. Taken together, our data indicates that Bacillus cereus AR156 pretreatment primes ISR to Pst infection by suppressing miR825 and miR825* and activating the defense related genes they targeted.
Project description:Small RNAs play an important role in plant innate immunity. However, their regulatory function in induced systemic resistance (ISR) triggered by plant growth-promoting rhizobacteria remains unclear. Here, using Arabidopsis as a model system, one plant endogenous small RNA, miR472, was identified as an important regulator involved in the process of Bacillus cereus AR156 ISR against Pseudomonas syringae pv. tomato (Pst) DC3000. The results revealed that miR472 was down-regulated with B. cereus AR156 treatment by comparing small RNA profiles and northern blot analysis of Arabidopsis with or without B. cereus AR156 treatment. Plants overexpressing miR472 showed higher susceptibility to Pst DC3000; by contrast, plant lines with miR472 knocked down/out showed the opposite. The transcriptome sequencing revealed thousands of differentially expressed genes in the transgenic plants. Target prediction showed that miR472 targets lots of coiled coil nucleotide-binding site (NBS) and leucine-rich repeat (LRR) type resistance genes and the expression of these targets was negatively correlated with the expression of miR472. In addition, transgenic plants with knocked-out target genes exhibited decreased resistance to Pst DC3000 invasion. Quantitative reverse transcription PCR results indicated that target genes of miR472 were expressed during the process of B. cereus AR156-triggered ISR. Taken together, our results demonstrate that the miR472-mediated silencing pathway is an important regulatory checkpoint occurring via post-transcriptional control of NBS-LRR genes during B. cereus AR156-triggered ISR in Arabidopsis.
Project description:Induced resistance response is a potent and cost effective plant defense against pathogen attack. The effectiveness and underlying mechanisms of the suppressive ability by Bacillus cereus AR156 to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) in Arabidopsis has been investigated previously; however, the strength of induced systemic resistance (ISR) activity against Botrytis cinerea remains unknown. Here, we show that root-drench application of AR156 significantly reduces disease incidence through activation of ISR. This protection is accompanied with multilayered ISR defense response activated via enhanced accumulation of PR1 protein expression in a timely manner, hydrogen peroxide accumulation and callose deposition, which is significantly more intense in plants with both AR156 pretreatment and B. cinerea inoculation than that in plants with pathogen inoculation only. Moreover, AR156 can trigger ISR in sid2-2 and NahG mutants, but not in jar1, ein2 and npr1 mutant plants. Our results indicate that AR156-induced ISR depends on JA/ET-signaling pathway and NPR1, but not SA. Also, AR156-treated plants are able to rapidly activate MAPK signaling and FRK1/WRKY53 gene expression, both of which are involved in pathogen associated molecular pattern (PAMP)-triggered immunity (PTI). The results indicate that AR156 can induce ISR by the JA/ET-signaling pathways in an NPR1-dependent manner and involves multiple PTI components.
Project description:Small RNAs function to regulate plant defense responses to pathogens. We previously showed that miR825 and miR825* downregulate Bacillus cereus AR156 (AR156)-triggered systemic resistance to Pseudomonassyringae pv. tomato DC3000 in Arabidopsis thaliana (Arabidopsis). Here, Northern blotting revealed that miR825 and miR825* were more strongly downregulated in wild type Arabidopsis Col-0 (Col-0) plants pretreated with AR156 than in nontreated plants upon Botrytis cinerea (B. cinerea) B1301 infection. Furthermore, compared with Col-0, transgenic plants with attenuated miR825 and miR825* expression were more resistant to B. cinerea B1301, yet miR825- and miR825*-overexpressing (OE) plants were more susceptible to the pathogen. With AR156 pretreatment, the transcription of four defense-related genes (PR1, PR2, PR5, and PDF1.2) and cellular defense responses (hydrogen peroxide production and callose deposition) were faster and stronger in miR825 and miR825* knockdown lines but weaker in their OE plants than in Col-0 plants upon pathogen attack. Also, AR156 pretreatment caused stronger phosphorylation of MPK3 and MPK6 and expression of FRK1 and WRKY53 genes upon B. cinerea B1301 inoculation in miR825 and miR825* knockdown plants than in Col-0 plants. Additionally, the assay of agrobacterium-mediated transient co-expression in Nicotiana benthamiana confirmed that AT5G40910, AT5G38850, AT3G04220, and AT5G44940 are target genes of miR825 or miR825*. Compared with Col-0, the target mutant lines showed higher susceptibility to B. cinerea B1301, while still expressing AR156-triggered induced systemic resistance (ISR). The two-way analysis of variance (ANOVA) revealed a significant (P < 0.01) interactive effect of treatment and genotype on the defense responses. Hence, miR825 and miR825*act as negative regulators of AR156-mediated systemic resistance to B. cinerea B1301 in Arabidopsis.
Project description:BACKGROUND:The adage from Shakespeare, "troubles, not as single spies, but in battalions come," holds true for Nicotiana attenuata, which is commonly attacked by both pathogens (Pseudomonas spp.) and herbivores (Manduca sexta) in its native habitats. Defense responses targeted against the pathogens can directly or indirectly influence the responses against the herbivores. Nadefensin is an effective induced defense gene against the bacterial pathogen Pseudomonas syringae pv tomato (PST DC3000), which is also elicited by attack from M. sexta larvae, but whether this defense protein influences M. sexta's growth and whether M. sexta-induced Nadefensin directly or indirectly influences PST DC3000 resistance are unknown. RESULTS:M. sexta larvae consumed less on WT and on Nadefensin-silenced N. attenuata plants that had previously been infected with PST DC3000 than on uninfected plants. WT plants infected with PST DC3000 showed enhanced resistance to PST DC3000 and decreased leaf consumption by M. sexta larvae, but larval mass gain was unaffected. PST DC3000-infected Nadefensin-silenced plants were less resistant to subsequent PST DC3000 challenge, and on these plants, M. sexta larvae consumed less and gained less mass. WT and Nadefensin-silenced plants previously damaged by M. sexta larvae were better able to resist subsequent PST DC3000 challenges than were undamaged plants. CONCLUSION:These results demonstrate that Na-defensin directly mediates defense against PST DC3000 and indirectly against M. sexta in N. attenuata. In plants that were previously infected with PST DC3000, the altered leaf chemistry in PST DC3000-resistant WT plants and PST DC3000-susceptible Nadefensin-silenced plants differentially reduced M. sexta's leaf consumption and mass gain. In plants that were previously damaged by M. sexta, the combined effect of the altered host plant chemistry and a broad spectrum of anti-herbivore induced metabolomic responses was more effective than Nadefensin alone in resisting PST DC3000.
Project description:Plant defense responses to biotic stresses are complex biological processes, all governed by sophisticated molecular regulations. Induced systemic resistance (ISR) is one of these defense mechanisms where beneficial bacteria or fungi prime plants to resist pathogens or pest attacks. In ISR, the defense arsenal in plants remains dormant and it is only triggered by an infection, allowing a better allocation of plant resources. Our group recently described that the well-known beneficial bacterium Paraburkholderia phytofirmans PsJN is able to induce Arabidopsis thaliana resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 through ISR, and that ethylene, jasmonate and salicylic acid are involved in this protection. Nevertheless, the molecular networks governing this beneficial interaction remain unknown. To tackle this issue, we analyzed the temporal changes in the transcriptome of PsJN-inoculated plants before and after being infected with Pst DC3000. These data were used to perform a gene network analysis to identify highly connected transcription factors. Before the pathogen challenge, the strain PsJN regulated 405 genes (corresponding to 1.8% of the analyzed genome). PsJN-inoculated plants presented a faster and stronger transcriptional response at 1-hour post infection (hpi) compared with the non-inoculated plants, which presented the highest transcriptional changes at 24 hpi. A principal component analysis showed that PsJN-induced plant responses to the pathogen could be differentiated from those induced by the pathogen itself. Forty-eight transcription factors were regulated by PsJN at 1 hpi, and a system biology analysis revealed a network with four clusters. Within these clusters LHY, WRKY28, MYB31 and RRTF1 are highly connected transcription factors, which could act as hub regulators in this interaction. Concordantly with our previous results, these clusters are related to jasmonate, ethylene, salicylic, acid and ROS pathways. These results indicate that a rapid and specific response of PsJN-inoculated plants to the virulent DC3000 strain could be the pivotal element in the protection mechanism.
Project description:Plants harbor various beneficial bacteria that modulate their innate immunity, resulting in induced systemic resistance (ISR) against various pathogens. However, the immune mechanisms underlying ISR triggered by Bacillus spp. and Pseudomonas spp. against pathogens with different lifestyles are not yet clearly elucidated. Here, we show that root drenching of Arabidopsis plants with Pseudomonas fluorescensPTA-CT2 and Bacillus subtilis PTA-271 can induce ISR against the necrotrophic fungus B. cinerea and the hemibiotrophic bacterium Pseudomonas syringae Pst DC3000. In the absence of pathogen infection, both beneficial bacteria do not induce any consistent change in systemic immune responses. However, ISR relies on priming faster and robust expression of marker genes for the salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) signaling pathways upon pathogen challenge. These responses are also associated with increased levels of SA, JA, and abscisic acid (ABA) in the leaves of bacterized plants after infection. The functional study also points at priming of the JA/ET and NPR1-dependent defenses as prioritized immune pathways in ISR induced by both beneficial bacteria against B. cinerea. However, B. subtilis-triggered ISR against Pst DC3000 is dependent on SA, JA/ET, and NPR1 pathways, whereas P. fluorescens-induced ISR requires JA/ET and NPR1 signaling pathways. The use of ABA-insensitive mutants also pointed out the crucial role of ABA signaling, but not ABA concentration, along with JA/ET signaling in primed systemic immunity by beneficial bacteria against Pst DC3000, but not against B. cinerea. These results clearly indicate that ISR is linked to priming plants for enhanced common and distinct immune pathways depending on the beneficial strain and the pathogen lifestyle.
Project description:<h4>Background</h4>Plants have evolved an array of constitutive and inducible defense strategies to restrict pathogen ingress. However, some pathogens still manage to invade plants and impair growth and productivity. Previous studies have revealed several key regulators of defense responses, and efforts have been made to use this information to develop disease resistant crop plants. These efforts are often hampered by the complexity of defense signaling pathways. To further elucidate the complexity of defense responses, we screened a population of T-DNA mutants in Colombia-0 background that displayed altered defense responses to virulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000).<h4>Results</h4>In this study, we demonstrated that the Arabidopsis Purple Acid Phosphatse5 (PAP5) gene, induced under prolonged phosphate (Pi) starvation, is required for maintaining basal resistance to certain pathogens. The expression of PAP5 was distinctly induced only under prolonged Pi starvation and during the early stage of Pst DC3000 infection (6 h.p.i). T-DNA tagged mutant pap5 displayed enhanced susceptibility to the virulent bacterial pathogen Pst DC3000. The pap5 mutation greatly reduced the expression of pathogen inducible gene PR1 compared to wild-type plants. Similarly, other defense related genes including ICS1 and PDF1.2 were impaired in pap5 plants. Moreover, application of BTH (an analog of SA) restored PR1 expression in pap5 plants.<h4>Conclusion</h4>Taken together, our results demonstrate the requirement of PAP5 for maintaining basal resistance against Pst DC3000. Furthermore, our results provide evidence that PAP5 acts upstream of SA accumulation to regulate the expression of other defense responsive genes. We also provide the first experimental evidence indicating the role PAP5 in plant defense responses.
Project description:The biological control process mediated by microbes relies on multiple interactions among plants, pathogens and biocontrol agents (BCAs). One such efficient BCA is Bacillus cereus AR156, a bacterial strain that controls a broad spectrum of plant diseases and potentially works as a microbe elicitor of plant immune reactions. It remains unclear, however, whether the interaction between plants and B. cereus AR156 may facilitate composition changes of plant root exudates and whether these changes directly affect the growth of both plant pathogens and B. cereus AR156 itself. Here, we addressed these questions by analyzing the influences of root exudate changes mediated by B. cereus AR156 during biocontrol against tomato bacterial wilt caused by Ralstonia solanacearum. Indeed, some upregulated metabolites in tomato root exudates induced by B. cereus AR156 (REB), such as lactic acid and hexanoic acid, induced the growth and motile ability of in vitro B. cereus AR156 cells. Exogenously applying hexanoic acid and lactic acid to tomato plants showed positive biocontrol efficacy (46.6 and 39.36%) against tomato bacterial wilt, compared with 51.02% by B. cereus AR156 itself. Furthermore, fructose, lactic acid, sucrose and threonine at specific concentrations stimulated the biofilm formation of B. cereus AR156 in Luria-Bertan- Glycerol- Magnesium medium (LBGM), and we also detected more colonized cells of B. cereus AR156 on the tomato root surface after adding these four compounds to the system. These observations suggest that the ability of B. cereus AR156 to induce some specific components in plant root exudates was probably involved in further biocontrol processes.
Project description:Bacillus amyloliquefaciens FZB42 is a type of plant growth-promoting rhizobacterium (PGPR) which activates induced systemic resistance (ISR) in Arabidopsis. Blocking of the synthesis of cyclic lipopeptides and 2,3-butanediol by FZB42, which have been demonstrated to be involved in the priming of ISR, results in the abolishment of the plant defence responses. To further clarify the ISR activated by PGPRs at the microRNA (miRNA) level, small RNA (sRNA) libraries from Arabidopsis leaves after root irrigation with FZB42, FZB42?sfp?alsS and control were constructed and sequenced. After fold change selection, promoter analysis and target prediction, miR846-5p and miR846-3p from the same precursor were selected as candidate ISR-associated miRNAs. miR846 belongs to the non-conserved miRNAs, specifically exists in Arabidopsis and its function in the plant defence response remains unclear. The disease severity of transgenic Arabidopsis overexpressing miR846 (OEmiR846) or knockdown miR846 (STTM846) against Pseudomonas syringae DC3000 suggests that the miR846 expression level in Arabidopsis is negatively correlated with disease resistance. Moreover, miR846 in Arabidopsis Col-0 is repressed after methyl jasmonate treatment. In addition, jasmonic acid (JA) signalling-related genes are up-regulated in STTM846, and the stomatal apertures of STTM846 are also less than those in Arabidopsis Col-0 after methyl jasmonate treatment. Furthermore, the disease resistance of STTM846 transgenic Arabidopsis against Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is blocked by the addition of the JA biosynthetic inhibitor diethyldiethiocarbamic acid (DIECA). Taken together, our results suggest that B. amyloliquefaciens FZB42 inoculation suppresses miR846 expression to induce Arabidopsis systemic resistance via a JA-dependent signalling pathway.