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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.

The kiwifruit bacterial canker (Pseudomonas syringae pv. actinidiae; Psa) causes severe damage to kiwifruit production worldwide. Psa biovar 6 (Psa6), which was isolated in Japan in 2015, produces two types of phytotoxins: coronatine and phaseolotoxin. To elucidate the unique virulence of Psa6, we performed transcriptomic analysis of phytotoxin synthesis genes and type III effector genes in in vitro cultivation using various media. The genes related to phytotoxin synthesis and effectors of Psa6 were strictly regulated in the coronatine-inducing mediums (HS and HSC); 14 of 23 effector genes and a hrpL sigma factor gene were induced at 3 h after transferring to the media (early-inducible genes), and phytotoxin synthesis genes such as argD of phaseolotoxin and cfl of coronatine were induced at 6 and 12 h after transferring to the media (late-inducible genes). In contrast, induction of these genes was not observed in the hrp-inducing medium. Next, to examine whether the changes in gene expression in different media is specific to Psa6, we investigated gene expression in other related bacteria. For Psa biovar 1 (Psa1), biovar 3 (Psa3), and P. s. pv. glycinea (Psg), no clear trends were observed in expression behavior across various culture media and incubation times. Therefore, Psa6 seems to exert its virulence efficiently by using two phytotoxins and effectors according to environmental changes. This is not seen in other biovars and pathovars, so it is thought that Psa6 has acquired its own balance of virulence.

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 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.

Pseudomonas syringae is the most widespread bacterial pathogen in plants. Several strains of P. syringae produce a phytotoxin, coronatine (COR), which acts as a jasmonic acid mimic and inhibits plant defense responses and contributes to disease symptom development. In this study, we found that COR inhibits early defense responses during nonhost disease resistance. Stomatal closure induced by a nonhost pathogen, P. syringae pv. tabaci, was disrupted by COR in tomato epidermal peels. In addition, nonhost HR cell death triggered by P. syringae pv. tabaci on tomato was remarkably delayed when COR was supplemented along with P. syringae pv. tabaci inoculation. Using isochorismate synthase (ICS)-silenced tomato plants and transcript profiles of genes in SA- and JA-related defense pathways, we show that COR suppresses SA-mediated defense during nonhost resistance.