Pseudomonas cannabina pv. alisalensis Virulence Factors Are Involved in Resistance to Plant-Derived Antimicrobials during Infection

Bacteria are exposed to and tolerate diverse and potentially toxic compounds in the natural environment. While efflux transporters are generally thought to involve bacterial antibiotic resistance in vitro, their contributions to plant bacterial virulence have so far been poorly understood. Pseudomonas cannabina pv. alisalensis (Pcal) is a causal agent of bacterial blight of Brassicaceae. We here demonstrated that NU19, which is mutated in the resistance-nodulation-cell division (RND) transporter encoded gene, showed reduced virulence on cabbage compared to WT, indicating that the RND transporter contributes to Pcal virulence on cabbage. We also demonstrated that brassinin biosynthesis was induced after Pcal infection. Additionally, the RND transporter was involved in resistance to plant-derived antimicrobials and antibiotics, including the cabbage phytoalexin brassinin. These results suggest that the RND transporter extrudes plant-derived antimicrobials and contributes to Pcal virulence. We also found that the RND transporter contributes to Pcal virulence on Brassicaceae and tomato, but not on oat. These results suggest that the RND transporter contributes to Pcal virulence differentially depending on the host-plant species. Lastly, our expression-profile analysis indicated that the type-three secretion system (TTSS), which is essential for pathogenesis, is also involved in suppressing brassinin biosynthesis. Taken together, our results suggest that several Pcal virulence factors are involved in resistance to plant-derived antimicrobials and bacterial survival during infection

<it>Arabidopsis </it>seedling flood-inoculation technique: a rapid and reliable assay for studying plant-bacterial interactions

<p>Abstract</p> <p>Background</p> <p>The <it>Arabidopsis thaliana-Pseudomonas syringae </it>model pathosystem is one of the most widely used systems to understand the mechanisms of microbial pathogenesis and plant innate immunity. Several inoculation methods have been used to study plant-pathogen interactions in this model system. However, none of the methods reported to date are similar to those occurring in nature and amicable to large-scale mutant screens.</p> <p>Results</p> <p>In this study, we developed a rapid and reliable seedling flood-inoculation method based on young <it>Arabidopsis </it>seedlings grown on MS medium. This method has several advantages over conventional soil-grown plant inoculation assays, including a shorter growth and incubation period, ease of inoculation and handling, uniform infection and disease development, requires less growth chamber space and is suitable for high-throughput screens. In this study we demonstrated the efficacy of the <it>Arabidopsis </it>seedling assay to study 1) the virulence factors of <it>P. syringae </it>pv. <it>tomato </it>DC3000, including type III protein secretion system (TTSS) and phytotoxin coronatine (COR); 2) the effector-triggered immunity; and 3) <it>Arabidopsis </it>mutants affected in salicylic acid (SA)- and pathogen-associated molecular pattern (PAMPs)-mediated pathways. Furthermore, we applied this technique to study nonhost resistance (NHR) responses in <it>Arabidopsis </it>using nonhost pathogens, such as <it>P. syringae </it>pv. <it>tabaci</it>, pv. <it>glycinea </it>and pv. <it>tomato </it>T1, and confirmed the functional role of FLAGELLIN-SENSING 2 (FLS2) in NHR.</p> <p>Conclusions</p> <p>The <it>Arabidopsis </it>seedling flood-inoculation assay provides a rapid, efficient and economical method for studying <it>Arabidopsis-Pseudomonas </it>interactions with minimal growth chamber space and time. This assay could also provide an excellent system for investigating the virulence mechanisms of <it>P. syringae</it>. Using this method, we demonstrated that FLS2 plays a critical role in conferring NHR against nonhost pathovars of <it>P. syringae</it>, but not to <it>Xanthomonas campestris </it>pv. <it>vesicatoria</it>. This method is potentially ideal for high-throughput screening of both <it>Arabidopsis </it>and pathogen mutants.</p

NADPH-dependent thioredoxin reductase C plays a role in nonhost disease resistance against Pseudomonas syringae pathogens by regulating chloroplast-generated reactive oxygen species

Chloroplasts are cytoplasmic organelles for photosynthesis in eukaryotic cells. In addition, recent studies have shown that chloroplasts have a critical role in plant innate immunity against invading pathogens. Hydrogen peroxide is a toxic by-product from photosynthesis, which also functions as a signaling compound in plant innate immunity. Therefore, it is important to regulate the level of hydrogen peroxide in response to pathogens. Chloroplasts maintain components of the redox detoxification system including enzymes such as 2-Cys peroxiredoxins (2-Cys Prxs), and NADPH-dependent thioredoxin reductase C (NTRC). However, the significance of 2-Cys Prxs and NTRC in the molecular basis of nonhost disease resistance is largely unknown. We evaluated the roles of Prxs and NTRC using knock-out mutants of Arabidopsis in response to nonhost Pseudomonas syringae pathogens. Plants lacking functional NTRC showed localized cell death (LCD) accompanied by the elevated accumulation of hydrogen peroxide in response to nonhost pathogens. Interestingly, the Arabidopsis ntrc mutant showed enhanced bacterial growth and disease susceptibility of nonhost pathogens. Furthermore, the expression profiles of the salicylic acid (SA) and jasmonic acid (JA)-mediated signaling pathways and phytohormone analyses including SA and JA revealed that the Arabidopsis ntrc mutant shows elevated JA-mediated signaling pathways in response to nonhost pathogen. These results suggest the critical role of NTRC in plant innate immunity against nonhost P. syringae pathogens

Large–Scale Transposon Mutagenesis Reveals Type III Secretion Effector HopR1 Is a Major Virulence Factor in <i>Pseudomonas syringae</i> pv. <i>actinidiae</i>

Bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa) is a serious threat to kiwifruit production worldwide. Four biovars (Psa biovar 1; Psa1, Psa biovar 3; Psa3, Psa biovar 5; Psa5, and Psa biovar 6; Psa6) were reported in Japan, and virulent Psa3 strains spread rapidly to kiwifruit production areas worldwide. Therefore, there is an urgent need to develop critical management strategies for bacterial canker based on dissecting the dynamic interactions between Psa and kiwifruit. To investigate the molecular mechanism of Psa3 infection, we developed a rapid and reliable high-throughput flood-inoculation method using kiwifruit seedlings. Using this inoculation method, we screened 3000 Psa3 transposon insertion mutants and identified 91 reduced virulence mutants and characterized the transposon insertion sites in these mutants. We identified seven type III secretion system mutants, and four type III secretion effectors mutants including hopR1. Mature kiwifruit leaves spray-inoculated with the hopR1 mutant showed significantly reduced virulence compared to Psa3 wild-type, indicating that HopR1 has a critical role in Psa3 virulence. Deletion mutants of hopR1 in Psa1, Psa3, Psa5, and Psa6 revealed that the type III secretion effector HopR1 is a major virulence factor in these biovars. Moreover, hopR1 mutants of Psa3 failed to reopen stomata on kiwifruit leaves, suggesting that HopR1 facilitates Psa entry through stomata into plants. Furthermore, defense related genes were highly expressed in kiwifruit plants inoculated with hopR1 mutant compared to Psa wild-type, indicating that HopR1 suppresses defense-related genes of kiwifruit. These results suggest that HopR1 universally contributes to virulence in all Psa biovars by overcoming not only stomatal-based defense, but also apoplastic defense

Jasmonate ZIM-Domain (JAZ) Protein Regulates Host and Nonhost Pathogen-Induced Cell Death in Tomato and <i>Nicotiana benthamiana</i>

<div><p>The nonhost-specific phytotoxin coronatine (COR) produced by several pathovars of <i>Pseudomonas syringae</i> functions as a jasmonic acid-isoleucine (JA-Ile) mimic and contributes to disease development by suppressing plant defense responses and inducing reactive oxygen species in chloroplast. It has been shown that the F-box protein CORONATINE INSENSITIVE 1 (COI1) is the receptor for COR and JA-Ile. JASMONATE ZIM DOMAIN (JAZ) proteins act as negative regulators for JA signaling in <i>Arabidopsis</i>. However, the physiological significance of JAZ proteins in <i>P. syringae</i> disease development and nonhost pathogen-induced hypersensitive response (HR) cell death is not completely understood. In this study, we identified <i>JAZ</i> genes from tomato, a host plant for <i>P. syringae</i> pv. <i>tomato</i> DC3000 (<i>Pst</i> DC3000), and examined their expression profiles in response to COR and pathogens. Most <i>JAZ</i> genes were induced by COR treatment or inoculation with COR-producing <i>Pst</i> DC3000, but not by the COR-defective mutant DB29. Tomato SlJAZ2, SlJAZ6 and SlJAZ7 interacted with SlCOI1 in a COR-dependent manner. Using virus-induced gene silencing (VIGS), we demonstrated that SlJAZ2, SlJAZ6 and SlJAZ7 have no effect on COR-induced chlorosis in tomato and <i>Nicotiana benthamiana</i>. However, <i>SlJAZ2</i>-, <i>SlJAZ6</i>- and <i>SlJAZ7</i>-silenced tomato plants showed enhanced disease-associated cell death to <i>Pst</i> DC3000. Furthermore, we found delayed HR cell death in response to the nonhost pathogen <i>Pst</i> T1 or a pathogen-associated molecular pattern (PAMP), INF1, in <i>SlJAZ2</i>- and <i>SlJAZ6</i>-silenced <i>N. benthamiana</i>. These results suggest that tomato JAZ proteins regulate the progression of cell death during host and nonhost interactions.</p></div

COR-dependent interaction between SlCOI1 and SlJAZs.

<p>(<b>A</b>) Interaction between SlCOI1 and SlJAZs. (<b>B</b>) Interaction between SlNINJA and SlJAZs. The MaV203 yeast strain carrying the construct for SlCOI1 (bait) and SlJAZs (prey) was grown on synthetic dropout (SD) glucose medium without Leu, Trp and His in the absence or presence of COR (20 µM). Photographs were taken at 5 days.</p

Expression profiles of <i>JAZ</i> genes in tomato seedlings inoculated with <i>P. syringae</i> pv. <i>tomato</i> DC3000 (<i>Pst</i> DC3K) or the COR-defective mutant <i>Pst</i> DB29, or treated with COR.

<p>Tomato seedlings were treated with distilled water (mock control), COR (20 pmol) or inoculated with <i>Pst</i> DC3K or <i>Pst</i> DB29. The expression of genes encoding tomato JAZs was evaluated by real-time quantitative RT-PCR. Bars represent the means ± standard deviation (SD).</p

Involvement of SGT1 in COR-mediated signal transduction pathway leading to disease symptom development

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), that causes bacterial speck disease on tomato, produces a non-host-specific virulence effector, coronatine (COR). COR functions as a jasmonic acid (JA)-isoleucine mimic in planta and has multiple roles in the pathogenicity of Pst DC3000. One of the hallmarks of bacterial speck disease on tomato is the formation of necrotic lesions surrounded by chlorosis and COR is required for disease development. However, the molecular basis of COR-mediated disease symptom development including chlorosis and necrosis is still largely unknown. In our recent publication in New Phytologist, using virus-induced gene silencing (VIGS) based reverse genetics screen, we demonstrated that SGT1 (suppressor of G2 allele of skp1) is required for COR-induced chlorosis in Nicotiana benthamiana. SGT1-silenced tomato leaves showed a complete loss of COR-induced chlorosis and reduced disease symptom development after the inoculation with Pst DC3000. Furthermore, Arabidopsis sgt1b mutant was less sensitive to COR-induced root growth inhibition and showed delayed Pst DC3000 disease symptoms. In this addendum, we discuss the possible contribution of SGT1 to COR-mediated signal transduction pathway leading to disease symptom development during Pst DC3000 pathogenesis in tomato and Arabidopsis