HexR Transcription Factor Contributes to Pseudomonas cannabina pv. alisalensis Virulence by Coordinating Type Three Secretion System Genes.

Pseudomonas cannabina pv. alisalensis (Pcal) causes bacterial blight on cabbage. We previously conducted a screening for reduced virulence using Tn5 transposon mutants and identified one of the transcriptional factors, HexR, as a potential Pcal virulence factor. However, the role of HexR in plant pathogenic Pseudomonas virulence has not been investigated well. Here, we demonstrated that the Pcal hexR mutant showed reduced disease symptoms and bacterial populations on cabbage, indicating that HexR contributes to Pcal virulence. We used RNA-seq analysis to characterize the genes regulated by HexR. We found that several type three secretion system (T3SS)-related genes had lower expression of the Pcal hexR mutant. Five genes were related to T3SS machinery, two genes were related to type three helper proteins, and three genes encoded type three effectors (T3Es). We also confirmed that T3SS-related genes, including hrpL, avrPto, hopM1, and avrE1, were also down-regulated in the Pcal hexR mutant both in culture and in vivo by using RT-qPCR. T3SS functions to suppress plant defense in host plants and induce hypersensitive response (HR) cell death in non-host plants. Therefore, we investigated the expression profiles of cabbage defense-related genes, including PR1 and PR5, and found that the expressions of these genes were greater in the Pcal hexR mutant. We also demonstrated that the hexR mutant did not induce HR cell death in non-host plants, indicating that HexR contributes in causing HR in nonhost plants. Together, these results indicate that the mutation in hexR leads to a reduction in the T3SS-related gene expression and thus an impairment in plant defense suppression, reducing Pcal virulence.

Prevention of Stomatal Entry as a Strategy for Plant Disease Control against Foliar Pathogenic Pseudomonas Species.

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.

Controlling stomatal aperture, a potential strategy for managing plant bacterial disease.

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. As a results, we 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. Indeed, Cys, Glu, and Lys, which showed a protective effect on cabbage, reduced stomatal aperture width and bacterial entry. Therefore, managing stomatal aperture can be a powerful strategy for controlling bacterial disease.

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.

Functional diversity of Medicago truncatula RNA polymerase II CTD phosphatase isoforms produced in the Arabidopsis thaliana superexpression platform.

Medicago truncatula is a model system for legume plants, which has substantially expanded the genome relative to the prototypical model dicot plant, Arabidopsis thaliana. An essential transcriptional regulator, FCP1 (transcription factor IIF-interacting RNA polymerase II carboxyl-terminal phosphatase 1) ortholog, is encoded by a single essential gene CPL4 (CTD-phosphatase-like 4), whereas M. truncatula genome contains four genes homologous to FCP1/AtCPL4, and splicing variants of MtCPL4 are observed. Functional diversification of MtCPL4 family proteins was analyzed using recombinant proteins (MtCPL4a1, MtCPL4a2, and MtCPL4b) produced in Arabidopsis cell culture system developed for plant protein overexpression. In vitro CTD phosphatase assay using highly purified MtCPL4 preparations revealed a potent CTD phosphatase activity in MtCPL4b, but not two splicing variants of MtCPL4a. On the other hand, in planta binding assay to RNA polymerase II (pol II) revealed a greater pol II-binding activity of both MtCPL4a variants. Our results indicate functional diversification of MtCPL4 isoforms and suggest the presence of a large number of functionally specialized CTD-phosphatase-like proteins in plants.

Colletotrichum orbiculare strains distributed in Japan: race identification and evaluation of virulence to cucurbits.

The pathogen Colletotrichum orbiculare is causal fungus of cucurbit anthracnose. Multiple races have been identified in the United States, suggesting that it is necessary to cultivate suitable resistant cultivars and breed new cultivars with the most suitable resistance gene. This study examined the pathogenicity and virulence of 20 strains in Japan to clarify the existence of races and virulence differences. Based on the symptoms on inoculated cotyledons and true leaves of watermelon, we could evaluate the compatibility of each strain to each host cultivar. Our analysis based on the reaction to the host cultivar harboring the resistance gene Ar-1 (Cla001017) revealed the existence of three races in Japan. An alarming result was that a race that overcame Ar-1, which is a target gene in current watermelon breeding in Japan, is present in Japan. The cucumber and melon host cultivars showed diverse symptoms, whereas a squash cultivar was resistant to all strains. Three strains caused severe damage even to the most resistant cucumber cultivar 'Ban Kyuri' and resistant cultivars harboring Cssgr, a well-known gene conferring loss-of-susceptibility resistance. Screening genetic resources for novel resistance genes using strains with high virulence is of vital importance for watermelon, cucumber, and melon production.

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.

Multiple virulence factors regulated by AlgU contribute to the pathogenicity of Pseudomonas savastanoi pv. glycinea in soybean.

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.

Covering Soybean Leaves With Cellulose Nanofiber Changes Leaf Surface Hydrophobicity and Confers Resistance Against Phakopsora pachyrhizi.

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 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.

Pseudmonas cannabina pv. alisalensis TrpA Is Required for Virulence in Multiple Host Plants.

Pseudomonas cannabina pv. alisalensis (Pcal) causes bacterial leaf spot and blight of Brassicaceae and Poaceae. We previously identified several potential Pcal virulence factors with transposon mutagenesis. Among these a trpA mutant disrupted the tryptophan synthase alpha chain, and had an effect on disease symptom development and bacterial multiplication. To assess the importance of TrpA in Pcal virulence, we characterized the trpA mutant based on inoculation test and Pcal gene expression profiles. The trpA mutant showed reduced virulence when dip- and syringe-inoculated on cabbage and oat. Moreover, epiphytic bacterial populations of the trpA mutant were also reduced compared to the wild-type (WT). These results suggest that TrpA contributes to bacterial multiplication on the leaf surface and in the apoplast, and disease development. Additionally, several Brassicaceae (including Japanese radish, broccoli, and Chinese cabbage) also exhibited reduced symptom development when inoculated with the trpA mutant. Moreover, trpA disruption led to downregulation of bacterial virulence genes, including type three effectors (T3Es) and the phytotoxin coronatine (COR), and to upregulation of tryptophan biosynthesis genes. These results indicate that a trade-off between virulence factor production and Pcal multiplication with tryptophan might be regulated in the infection processes.

Coronatine Contributes to Pseudomonas cannabina pv. alisalensis Virulence by Overcoming Both Stomatal and Apoplastic Defenses in Dicot and Monocot Plants.

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.[Formula: see text] 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.

Acibenzolar-S-Methyl Activates Stomatal-Based Defense Systemically in Japanese Radish.

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.

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.

Transposon mutagenesis reveals Pseudomonas cannabina pv. alisalensis optimizes its virulence factors for pathogenicity on different hosts.

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.

Soybean leaves transcriptomic data dissects the phenylpropanoid pathway genes as a defence response against Phakopsora pachyrhizi.

Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, is responsible for severe yield losses of up to 90% in all soybean producing countries. Till today, eight resistance to Phakopsora pachyrhizi (Rpp) loci have been mapped in soybean. Their resistance mechanism is race specific but largely unknown. The transcriptomes of susceptible BRS184 and Rpp3 with ASR isolates T1-2 at 24 h after inoculation (hai) and without ASR inoculation (mock) were annotated by similarity searching with different databases. A total of 4518 differentially expressed genes were identified. We found 70.89%, 56.61%, 32.13%, and 56.04% genes in the protein family databases (PFAM), Gene Ontology (GO), Eukaryotic clusters of Orthologous Groups (KOG), and Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG), respectively. KEGG disclosed that 52% of the phenylpropanoid pathway related genes were up-regulated. The relative gene expression study for selected genes of that pathway was conducted by RT-qPCR using Rpp1-Rpp4 carrying lines with T1-2 infection. The RT-qPCR results revealed that the Rpp lines utilized these genes in a rate limiting manner as a defence response. With the exception of glycinol 4-dimethylallyltransferase (G4DT) and chalcone reductase (CHR), all the genes showed the greatest expression at 12 hai, but the gene expressions which occur between 24 and 96 hai make these Rpp lines unique to their respective ASR isolates. Moreover, functional coordination of arogenate dehydratase 6 (ADT6) and 4-hydroxy-3-methylbut-2-enyl diphosphate synthase (ispG), chalcone synthase (CHS) and CHR, and G4DT and phytyltransferase 3 (PT3) may have a great impact on soybean resistance against ASR.

Metabolic flux towards the (iso)flavonoid pathway in lignin modified alfalfa lines induces resistance against Fusarium oxysporum f. sp. medicaginis.

Downregulation of lignin in alfalfa (Medicago sativa L.) is associated with increased availability of cell wall polysaccharides in plant cells. We tested transgenic alfalfa plants downregulated for Caffeoyl-CoA O-methyltransferase (CCoAOMT) against an economically important fungal disease of alfalfa, Fusarium wilt caused by Fusarium oxysporum f. sp. medicaginis, and found it more resistant to this disease. Transcriptomic and metabolomic analyses indicated that the improved disease resistance against Fusarium wilt is due to increased accumulation and/or spillover of flux towards the (iso)flavonoid pathway. Some (iso)flavonoids and their pathway intermediate compounds showed strong accumulation in CCoAOMT downregulated plants after F. oxysporum f. sp. medicaginis inoculation. The identified (iso)flavonoids, including medicarpin and 7,4'-dihydroxyflavone, inhibited the in vitro growth of F. oxysporum f. sp. medicaginis. These results suggested that the increased accumulation and/or shift/spillover of flux towards the (iso)flavonoid pathway in CCoAOMT downregulated plants is associated with induced disease resistance.

The small GTPase, nucleolar GTP-binding protein 1 (NOG1), has a novel role in plant innate immunity.

Plant defense responses at stomata and apoplast are the most important early events during plant-bacteria interactions. The key components for the signaling of stomatal defense and nonhost resistance have not been fully characterized. Here we report the newly identified small GTPase, Nucleolar GTP-binding protein 1 (NOG1), functions for plant immunity against bacterial pathogens. Virus-induced gene silencing of NOG1 compromised nonhost resistance in N. benthamiana and tomato. Comparative genomic analysis showed that two NOG1 copies are present in all known plant species: NOG1-1 and NOG1-2. Gene downregulation and overexpression studies of NOG1-1 and NOG1-2 in Arabidopsis revealed the novel function of these genes in nonhost resistance and stomatal defense against bacterial pathogens, respectively. Specially, NOG1-2 regulates guard cell signaling in response to biotic and abiotic stimuli through jasmonic acid (JA)- and abscisic acid (ABA)-mediated pathways. The results here provide valuable information on the new functional role of small GTPase, NOG1, in guard cell signaling and early plant defense in response to bacterial pathogens.

The SAL-PAP Chloroplast Retrograde Pathway Contributes to Plant Immunity by Regulating Glucosinolate Pathway and Phytohormone Signaling.

Chloroplasts have a crucial role in plant immunity against pathogens. Increasing evidence suggests that phytopathogens target chloroplast homeostasis as a pathogenicity mechanism. In order to regulate the performance of chloroplasts under stress conditions, chloroplasts produce retrograde signals to alter nuclear gene expression. Many signals for the chloroplast retrograde pathway have been identified, including chlorophyll intermediates, reactive oxygen species, and metabolic retrograde signals. Although there is a reasonably good understanding of chloroplast retrograde signaling in plant immunity, some signals are not well-understood. In order to understand the role of chloroplast retrograde signaling in plant immunity, we investigated Arabidopsis chloroplast retrograde signaling mutants in response to pathogen inoculation. sal1 mutants (fry1-2 and alx8) responsible for the SAL1-PAP retrograde signaling pathway showed enhanced disease symptoms not only to the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000 but, also, to the necrotrophic pathogen Pectobacterium carotovorum subsp. carotovorum EC1. Glucosinolate profiles demonstrated the reduced accumulation of aliphatic glucosinolates in the fry1-2 and alx8 mutants compared with the wild-type Col-0 in response to DC3000 infection. In addition, quantification of multiple phytohormones and analyses of their gene expression profiles revealed that both the salicylic acid (SA)- and jasmonic acid (JA)-mediated signaling pathways were down-regulated in the fry1-2 and alx8 mutants. These results suggest that the SAL1-PAP chloroplast retrograde pathway is involved in plant immunity by regulating the SA- and JA-mediated signaling pathways.

Pseudomonas syringae Flood-inoculation Method in Arabidopsis.

Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), which causes bacterial speck disease of tomato, has been used as a model pathogen because of its pathogenicity on Arabidopsis thaliana. Here, we demonstrate a rapid and reliable flood-inoculation method based on young Arabidopsis seedlings grown on one-half strength MS medium. We also describe a method to evaluate the bacterial growth in Arabidopsis.