Pseudomonas cannabina pv. alisalensis is a causative agent of bacterial blight of crucifers including cabbage, radish, and broccoli. Importantly, P. cannabina pv. alisalensis can infect not only a wide range of Brassicaceae spp. but, also, green manure crops such as oat. However, P. cannabina pv. alisalensis virulence mechanisms have not been investigated and are not fully understood. We focused on coronatine (COR) function, which is one of the well-known P. syringae pv. tomato DC3000 virulence factors, in P. cannabina pv. alisalensis infection processes on both dicot and monocot plants. Cabbage and oat plants dip-inoculated with a P. cannabina pv. alisalensis KB211 COR mutant (ΔcmaA) exhibited reduced virulence compared with P. cannabina pv. alisalensis wild type (WT). Moreover, ΔcmaA failed to reopen stomata on both cabbage and oat, suggesting that COR facilitates P. cannabina pv. alisalensis entry through stomata into both plants. Furthermore, cabbage and oat plants syringe-infiltrated with ΔcmaA also showed reduced virulence, suggesting that COR is involved in overcoming not only stomatal-based defense but also apoplastic defense. Indeed, defense-related genes, including PR1 and PR2, were highly expressed in plants inoculated with ΔcmaA compared with WT, indicating that COR suppresses defense-related genes of both cabbage and oat. Additionally, salicylic acid accumulation increases after ΔcmaA inoculation compared with WT. Taken together, COR contributes to causing disease by suppressing stomatal-based defense and apoplastic defense in both dicot and monocot plants. Here, we investigated COR functions in the interaction of P. cannabina pv. alisalensis and different host plants (dicot and monocot plants), using genetically and biochemically defined COR deletion mutants. [ ] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2021.

The genus Pseudomonas includes some of the most problematic and studied foliar bacterial pathogens. Generally, in a successful disease cycle there is an initial epiphytic lifestyle on the leaf surface and a subsequent aggressive endophytic stage inside the leaf apoplast. Leaf-associated bacterial pathogens enter intercellular spaces and internal leaf tissues by natural surface opening sites, such as stomata. The stomatal crossing is complex and dynamic, and functional genomic studies have revealed several virulence factors required for plant entry. Currently, treatments with copper-containing compounds, where authorized and admitted, and antibiotics are commonly used against bacterial plant pathogens. However, strains resistant to these chemicals occur in the fields. Therefore, the demand for alternative control strategies has been increasing. This review summarizes efficient strategies to prevent bacterial entry. Virulence factors required for entering the leaf in plant-pathogenic Pseudomonas species are also discussed.

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.

Rhizoctonia solani is a basidiomycete phytopathogenic fungus that causes rapid necrosis in a wide range of crop species, leading to substantial agricultural losses worldwide. The species complex is divided into 13 anastomosis groups (AGs) based on hyphal fusion compatibility and further subdivided by culture morphology. While R. solani classifications were shown to be independent of host specificity, it remains unclear whether different R. solani isolates share similar virulence mechanisms. Here, we investigated the infectivity of Japanese R. solani isolates on Brachypodium distachyon and barley. Two isolates, AG-1 IA (from rice) and AG-4 HG-I+II (from cauliflower), infected leaves of both plants, but only AG-4 HG-I+II infected roots. B. distachyon accessions Bd3-1 and Gaz-4 and barley cultivar ‘Morex’ exhibited enhanced resistance to both isolates compared to B. distachyon Bd21 and barley cultivars ‘Haruna Nijo’ and ‘Golden Promise’. During AG-1 IA infection, but not AG-4 HG-I+II infection, resistant Bd3-1 and Morex induced genes for salicylic acid (SA) and N-hydroxypipecolic acid (NHP) biosynthesis. Pretreatment with SA or NHP conferred resistance to AG-1 IA, but not AG-4 HG-I+II, in susceptible B. distachyon Bd21 and barley Haruna Nijo. On the leaves of susceptible Bd21 and Haruna Nijo, AG-1 IA developed extensive mycelial networks with numerous infection cushions, which are specialized infection structures well-characterized in rice sheath blight. In contrast, AG-4 HG-I+II formed dispersed mycelial masses associated with underlying necrosis. We propose that the R. solani species complex encompasses at least two distinct infection strategies: AG-1 IA exhibits a hemibiotrophic lifestyle, while AG-4 HG-I+II follows a predominantly necrotrophic strategy.

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.