Large-Scale Transposon Mutagenesis Reveals Type III Secretion Effector HopR1 Is a Major Virulence Factor in Pseudomonas syringae pv. actinidiae.

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.

Phytotoxin synthesis genes and type III effector genes of Pseudomonas syringae pv. actinidiae biovar 6 are regulated by culture conditions.

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.

Flood inoculation of seedlings on culture medium to study interactions between Pseudomonas syringae pv. actinidiae and kiwifruit.

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.

Jasmonate ZIM-domain (JAZ) protein regulates host and nonhost pathogen-induced cell death in tomato and Nicotiana benthamiana.

The nonhost-specific phytotoxin coronatine (COR) produced by several pathovars of Pseudomonas syringae 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 Arabidopsis. However, the physiological significance of JAZ proteins in P. syringae disease development and nonhost pathogen-induced hypersensitive response (HR) cell death is not completely understood. In this study, we identified JAZ genes from tomato, a host plant for P. syringae pv. tomato DC3000 (Pst DC3000), and examined their expression profiles in response to COR and pathogens. Most JAZ genes were induced by COR treatment or inoculation with COR-producing Pst 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 Nicotiana benthamiana. However, SlJAZ2-, SlJAZ6- and SlJAZ7-silenced tomato plants showed enhanced disease-associated cell death to Pst DC3000. Furthermore, we found delayed HR cell death in response to the nonhost pathogen Pst T1 or a pathogen-associated molecular pattern (PAMP), INF1, in SlJAZ2- and SlJAZ6-silenced N. benthamiana. These results suggest that tomato JAZ proteins regulate the progression of cell death during host and nonhost interactions.

Drought stress acclimation imparts tolerance to Sclerotinia sclerotiorum and Pseudomonas syringae in Nicotiana benthamiana.

Acclimation of plants with an abiotic stress can impart tolerance to some biotic stresses. Such a priming response has not been widely studied. In particular, little is known about enhanced defense capacity of drought stress acclimated plants to fungal and bacterial pathogens. Here we show that prior drought acclimation in Nicotiana benthamiana plants imparts tolerance to necrotrophic fungus, Sclerotinia sclerotiorum, and also to hemi-biotrophic bacterial pathogen, Pseudomonas syringae pv. tabaci. S. sclerotiorum inoculation on N. benthamiana plants acclimated with drought stress lead to less disease-induced cell death compared to non-acclimated plants. Furthermore, inoculation of P. syringae pv. tabaci on N. benthamiana plants acclimated to moderate drought stress showed reduced disease symptoms. The levels of reactive oxygen species (ROS) in drought acclimated plants were highly correlated with disease resistance. Further, in planta growth of GFPuv expressing P. syringae pv. tabaci on plants pre-treated with methyl viologen showed complete inhibition of bacterial growth. Taken together, these experimental results suggested a role for ROS generated during drought acclimation in imparting tolerance against S. sclerotiorum and P. syringae pv. tabaci. We speculate that the generation of ROS during drought acclimation primed a defense response in plants that subsequently caused the tolerance against the pathogens tested.

Coronatine inhibits stomatal closure and delays hypersensitive response cell death induced by nonhost bacterial 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.

NTRC and chloroplast-generated reactive oxygen species regulate Pseudomonas syringae pv. tomato disease development in tomato and Arabidopsis.

Coronatine (COR)-producing pathovars of Pseudomonas syringae, including pvs. tomato, maculicola, and glycinea, cause important diseases on tomato, crucifers, and soybean, respectively, and produce symptoms with necrotic lesions surrounded by chlorosis. The chlorosis is mainly attributed to COR. However, the significance of COR-induced chlorosis in localized lesion development and the molecular basis of disease-associated cell death is largely unknown. To identify host (chloroplast) genes that play a role in COR-mediated chlorosis, we used a forward genetics approach using Nicotiana benthamiana and virus-induced gene silencing and identified a gene which encodes 2-Cys peroxiredoxin (Prxs) that, when silenced, produced a spreading hypersensitive or necrosis-like phenotype instead of chlorosis after COR application in a COI1-dependent manner. Loss-of-function analysis of Prx and NADPH-dependent thioredoxin reductase C (NTRC), the central players of a chloroplast redox detoxification system, resulted in spreading accelerated P. syringae pv. tomato DC3000 disease-associated cell death with enhanced reactive oxygen species (ROS) accumulation in a COR-dependent manner in tomato and Arabidopsis. Consistent with these results, virulent strain DC3000 suppressed the expression of Prx and NTRC in Arabidopsis and tomato during pathogenesis. However, interestingly, authentic COR suppressed the expression of Prx and NTRC in tomato but not in Arabidopsis, suggesting that COR in conjunction with other effectors may modulate ROS and cell death in different host species. Taken together, these results indicated that NTRC or Prx function as a negative regulator of pathogen-induced cell death in the healthy tissues that surround the lesions, and COR-induced chloroplast-localized ROS play a role in enhancing the disease-associated cell death.

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.

SGT1 contributes to coronatine signaling and Pseudomonas syringae pv. tomato disease symptom development in tomato and Arabidopsis.

• 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 COR-induced 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 COR-induced chlorosis and subsequent necrotic disease development in tomato and Arabidopsis. SGT1 is therefore a component of the COR/JA-mediated signal transduction pathway.