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Bacterial brown stripe caused by Acidovorax avenae subsp. avenae (Aaa) causes serious damage on creeping bentgrass on golf course greens. However, there are few effective control strategies. Acibenzolar-S-methyl (ASM), a synthetic analog of salicylic acid (SA), has been used to protect plants from diseases by activating plant defense. Here, we demonstrated that bacterial populations were reduced at least 10 times after ASM pretreatment when plants were clip-inoculated with Aaa. In tests of ASM against Aaa on creeping bentgrass in golf courses, the preventive efficacy of ASM was 43–71% and curative efficacy was 57–86%. We also demonstrated that dip-inoculations of creeping bentgrass with Aaa caused disease and that pretreatment with ASM reduced bacterial populations. In a study of stomatal response after Aaa inoculation and ASM treatment, within 1 h, ASM systemically induced stomatal closure, which lasted for at least 1 week. These results suggest that ASM confers immediate, continuous stomatal closure in creeping bentgrass and provides powerful bacterial control in creeping bentgrass.

In efforts to control bacterial blight caused by Pseudomonas cannabina pv. alisalensis (Pcal) on cabbage using plant defense activators acibenzolar-S-methyl (ASM) and probenazole (PBZ), a soil drench with ASM and PBZ effectively suppressed Pcal lesion formation and reduced bacterial populations compared with a water drench. Although ASM and PBZ activated stomatal-based defense against Pcal resulting in lower bacterial populations and less-severe symptoms, ASM, but not PBZ, induced stomatal-based defense within 4 h after the soil drench. Thus, ASM and PBZ activate a stomatal-based defense in cabbage against Pcal but differ the timing of induction.

Bacterial blight of crucifers caused by Pseudomonas cannabina pv. alisalensis (Pcal) inflicts great damage on crucifer production. To explore efficient and sustainable strategies for Pcal disease control, we here investigated and screened for amino acids with reduced disease development. We found that exogenous foliar application with multiple amino acids reduced disease symptoms and bacterial populations in cabbage after spray-inoculation, but not syringe-inoculation. These results indicate that these amino acids showed a protective effect before Pcal entered plants. Therefore, we observed stomatal responses, which is a main gateway for Pcal entry into the apoplast, after amino acid treatments, and found several amino acids induce stomatal closure. Moreover, our findings demonstrated that reducing stomatal aperture width can limit bacterial entry into plants, leading to reduced disease symptoms. Therefore, managing stomatal aperture can be a new powerful strategy for controlling bacterial disease.

Pseudomonas savastanoi pv. glycinea (Psg) causes bacterial blight of soybean. To identify candidate virulence factors, transposon-mediated mutational analysis of Psg was carried out. We syringe-inoculated soybean leaves with Psg transposon mutants and identified 28 mutants which showed reduced virulence from 1,000 mutants screened. Next, we spray-inoculated soybean leaves with these mutants and demonstrated that the algU mutant showed significantly reduced virulence together with reduced bacterial populations in planta. Expression profiles comparison between the Psg wild-type (WT) and algU mutant in HSC broth revealed that expression of coronatine (COR)-related genes (including cmaA and corR) were down-regulated in the algU mutant compared with Psg WT. Moreover, we also showed that COR production were reduced in the algU mutant compared with WT. We also demonstrated that algD, which is related to alginate biosynthesis, showed reduced expression and biofilm formation was significantly suppressed in the algU mutant. Furthermore, hrpL also showed less expression in the algU mutant. These results indicate that AlgU plays a critical role in promoting Psg pathogenesis by regulating multiple virulence factors.

Bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa) is a serious threat to kiwifruit production. Highly virulent strains of Psa biovar3 (Psa3) have 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 interactions between Psa and kiwifruit. Here, we developed a rapid and reliable flood-inoculation method using kiwifruit seedlings grown on Murashige and Skoog medium. This method has several advantages over inoculation of conventional soil-grown plants. We demonstrated the utility of a kiwifruit seedling assay to study the virulence of Psa biovars and Psa3 virulence factors, including the type III secretion system (T3SS). Kiwifruit seedlings inoculated with Psa3 developed severe necrosis within 1 week, whereas those inoculated with a T3SS-deficient hrcN mutant of Psa3 did not. This method was also useful for analyzing expression profiles of genes involved in Psa3 virulence during infection, and revealed that the expression of genes encoding the T3SS and type III secreted effectors were strongly induced in planta. Our results indicate that the T3SS has an important role in Psa3 virulence, and the flood-inoculation assay using kiwifruit seedling is suitable for analyzing Psa and kiwifruit interactions.

Pseudomonas cannabina pv. alisalensis (Pcal), which causes bacterial blight of brassicaceous plants, is an economically important pathogen worldwide. Copper fungicide and antibiotics are major strategies to manage the disease caused by Pcal; however, a Pcal strain resistant to these chemicals has already been found, and severe outbreaks of bacterial blight have been reported on cabbage in Japan. Therefore, there is an urgent need to develop new Pcal management strategies. Plant defense activators could be useful not only to protect plants against invading pathogens, but also to reduce the amount of copper fungicides and antibiotics applied. However, the mechanisms by which plant defense activators contribute to controlling diseases remains unclear. In this work, we focused on cabbage and acibenzolar-S-methyl (ASM), a well-known plant defense activator. Expression profiles revealed that ASM induced expression of systemic acquired resistance (SAR) marker genes including PR1, PR2, and PR5 in cabbage plants. We also demonstrated that a soil drench with ASM 2 h before transplanting clearly reduced bacterial blight symptoms and reduced Pcal bacterial populations in cabbage. ASM application was also able to prime cabbage for Pcal resistance by activating stomatal-based defense. Our findings highlight that ASM protects plants from bacterial pathogens by activating stomatal-based defense.

Asian soybean rust (ASR) caused by Phakopsora pachyrhizi, an obligate biotrophic fungal pathogen, is the most devastating soybean production disease worldwide. Currently, timely fungicide application is the only means to control ASR in the field. We investigated cellulose nanofiber (CNF) application on ASR disease management. CNF-treated leaves showed reduced lesion number after P. pachyrhizi inoculation compared to control leaves, indicating that covering soybean leaves with CNF confers P. pachyrhizi resistance. We also demonstrated that formation of P. pachyrhizi pre-infection structures including germ-tubes and appressoria, and also gene expression related to these formations, such as chitin synthases (CHSs), were significantly suppressed in CNF-treated soybean leaves compared to control leaves. Moreover, contact angle measurement revealed that CNF converts soybean leaf surface properties from hydrophobic to hydrophilic. These results suggest that CNF can change soybean leaf surface hydrophobicity, conferring resistance against P. pachyrhizi, based on the reduced expression of CHSs, as well as reduced formation of pre-infection structures. This is the first study to investigate CNF application to control field disease.

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.

Acibenzolar-S-methyl (ASM) is a well-known plant activator, which is a synthetic analog of salicylic acid (SA). Recently, copper fungicides and antibiotics are major strategies for controlling bacterial diseases. However, resistant strains have already been found. Therefore, there is an increasing demand for sustainable new disease control strategies. We investigated the ASM disease control effect against Pseudomonas cannabina pv. alisalensis (Pcal), which causes bacterial blight on Japanese radish. In this study, we demonstrated that ASM effectively suppressed Pcal disease symptom development associated with reduced bacterial populations on Japanese radish leaves. Interestingly, we also demonstrated that ASM activated systemic acquired resistance (SAR), including stomatal-based defense on ASM-untreated upper and lower leaves. Reactive oxygen species (ROS) are essential second messengers in stomatal-based defense. We found that ASM induced stomatal closure by inducing ROS production through peroxidase. These results indicate that stomatal closure induced by ASM treatment is effective for preventing Pcal pathogen invasion into plants, and in turn reduction of disease development.

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), which causes bacterial speck disease of tomato, has been used as a model pathogen to investigate the molecular basis of plant–pathogen interactions. The function of many potential virulence factors encoded in the Pst DC3000 genome and their modes of action are not fully understood. P. syringae is known to produce the exopolysaccharide alginate. Although AlgU, a sigma factor, is known to regulate the expression of genes such as algD related to alginate biosynthesis, the molecular mechanisms of AlgU in the virulence of Pst DC3000 is still unclear. To investigate the function of AlgU and alginate in plant–bacterial pathogen interactions, we generated ΔalgU and ΔalgD mutants. After inoculation with ΔalgU but not ΔalgD, host plants of Pst DC3000 including tomato and Arabidopsis had milder disease symptoms and reduced bacterial populations. Expression profiles of Pst DC3000 genes revealed that AlgU can regulate not only the expression of genes encoding alginate biosynthesis, but also the expression of genes related to type III effectors and the phytotoxin coronatine (COR). We also demonstrated that the ΔalgU mutant showed full virulence in the Arabidopsis fls2 efr1 double mutant, which is compromised in the recognition of PAMPs. Further, the application of COR was able to restore the phenotype of the ΔalgU mutant in the stomatal response. These results suggest that AlgU has an important role in the virulence of Pst DC3000 by regulating COR production.

Pseudomonas cannabina pv. alisalensis (Pcal), which causes bacterial blight disease of Brassicaceae, is an economically important pathogen worldwide. To identify Pcal genes involved in pathogenesis, we conducted a screen for 1,040 individual Pcal KB211 Tn5 mutants with reduced virulence on cabbage plants using a dip-inoculation method. We isolated 53 reduced virulence mutants and identified several potential virulence factors involved in Pcal virulence mechanisms such as the type III secretion system, membrane transporters, transcription factors, and amino acid metabolism. Importantly, Pcal is pathogenic on a range of monocotyledonous and dicotyledonous plants. Therefore, we also carried out the inoculation test on oat plants, which are cultivated after cabbage cultivation as green manure crops. Interestingly among the 53 mutants, 31 mutants also exhibited reduced virulence on oat seedlings, indicating that Pcal optimizes its virulence factors for pathogenicity on different host plants. Our results highlight the importance of revealing the virulence factors for each plant host-bacterial interaction, and will provide new insights into Pcal virulence mechanisms.

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 ofArabidopsisin response to nonhostPseudomonas syringaepathogens. Plants lacking functional NTRC showed localized cell death (LCD) accompanied by the elevated accumulation of hydrogen peroxide in response to nonhost pathogens. Interestingly, theArabidopsis ntrcmutant 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 theArabidopsis ntrcmutant shows elevated JA-mediated signaling pathways in response to nonhost pathogen. These results suggest the critical role of NTRC in plant innate immunity against nonhostP. syringaepathogens.

Coronatine (COR) functions as a virulence factor in the interaction of Pseudomonas syringae pv. tomato strain DC3000 with tomato and Arabidopsis. COR consists of two moieties, coronafacic acid (CFA) and coronamic acid (CMA). Both COR and CFA function as structural and functional analogues of jasmonic acid (JA) and related signaling compounds such as methyl jasmonate (MeJA) and JA-isoleucine (JA-Ile). The precise function of COR and whether MeJA functions as an analogue of COR in disease development are not known. In this study, we analyzed whether the COR-defective mutant DB29, which is a CFA− CMA− mutant of DC3000, could be complemented for virulence via the exogenous application of COR, CFA, or MeJA. When tomato seedlings were inoculated with DB29 and supplemented with exogenous COR, the DB29 population multiplied in tomato seedlings and induced disease phenotypes similar to wild-type DC3000. Although the addition of exogenous MeJA or CFA enhanced the multiplication of DB29, wild-type disease phenotypes could not be restored with these compounds. Furthermore, inoculation of DB29 along with exogenous COR, but not MeJA or CFA, suppressed the expression of defense-related genes and increased the accumulation of reactive oxygen species, which are associated with severe chlorosis. Taken together, our results suggest that although COR targets the jasmonate pathway by mimicking JA, the function of COR in disease development is distinctly different from MeJA or CFA.

Background The Arabidopsis thaliana-Pseudomonas syringae 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. Results In this study, we developed a rapid and reliable seedling flood-inoculation method based on young Arabidopsis 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 Arabidopsis seedling assay to study 1) the virulence factors of P. syringae pv. tomato DC3000, including type III protein secretion system (TTSS) and phytotoxin coronatine (COR); 2) the effector-triggered immunity; and 3) Arabidopsis 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 Arabidopsis using nonhost pathogens, such as P. syringae pv. tabaci, pv. glycinea and pv. tomato T1, and confirmed the functional role of FLAGELLIN-SENSING 2 (FLS2) in NHR. Conclusions The Arabidopsis seedling flood-inoculation assay provides a rapid, efficient and economical method for studying Arabidopsis-Pseudomonas interactions with minimal growth chamber space and time. This assay could also provide an excellent system for investigating the virulence mechanisms of P. syringae. Using this method, we demonstrated that FLS2 plays a critical role in conferring NHR against nonhost pathovars of P. syringae, but not to Xanthomonas campestris pv. vesicatoria. This method is potentially ideal for high-throughput screening of both Arabidopsis and pathogen mutants.

International audience; Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) causes an economically important bacterial speck disease on tomato and produces symptoms with necrotic lesions surrounded by chlorosis. The chlorosis is mainly attributed to a jasmonic acid (JA)-isoleucine analogue, coronatine (COR), produced by Pst DC3000. However, the molecular processes underlying lesion development and COR-induced chlorosis are poorly understood. In this study, we took advantage of a chlorotic phenotype elicited by COR on Nicotiana benthamiana leaves and virus-induced gene silencing (VIGS) as a rapid reverse genetic screening tool and identified a role for SGT1 suppressor of G2 allele of skp1) in COR-induced chlorosis. Silencing of SGT1 in tomato resulted in reduction of disease-associated symptoms (cell death and chlorosis), suggesting a molecular connection between CORinduced chlorosis and cell death. In Arabidopsis, AtSGT1b but not AtSGT1a was required for COR responses, including root growth inhibition and Pst DC3000 symptom (water soaked lesion) development. Notably, overexpression of AtSGT1b did not alter Pst DC3000 symptoms or sensitivity to COR. Taken together, our results demonstrate that SGT1 ⁄ SGT1b is required for CORinduced chlorosis and subsequent necrotic disease development in tomato and Arabidopsis. SGT1 is therefore a component of the COR⁄ JA-mediated signal transduction pathway.

The phytotoxin coronatine (COR), which is produced by Pseudomonas syringae pv. tomato DC3000 (DC3000), has multiple roles in virulence that lead to chlorosis and a reduction in chlorophyll content. However, the physiological significance of COR-induced chlorosis in disease development is still largely unknown. Global expression analysis demonstrated that DC3000 and COR, but not the COR-defective mutant DB29, caused reduced expression of photosynthesis-related genes and result in a 1.5- to 2-fold reduction in maximum quantum efficiency of photosystem II (F(V)/F(M)). Tomato (Solanum lycopersicum) seedlings inoculated with DC3000 and incubated in a long daily photoperiod showed more necrosis than inoculated seedlings incubated in either dark or a short daily photoperiod. The accumulation of reactive oxygen species (ROS) was detected in cotyledons inoculated with either purified COR or DC3000 but not in tissues inoculated with DB29. Interestingly, COR-induced ROS accumulated only in light and was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and diphenylene iodonium, which function to inhibit electron transport from PSII. Furthermore, COR and DC3000 suppressed expression of the gene encoding the thylakoid Cu/Zn superoxide dismutase but not the cytosolic form of the same enzyme. In conclusion, these results demonstrate a role for COR-induced effects on photosynthetic machinery and ROS in modulating necrotic cell death during bacterial speck disease of tomato.

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.