Hypersensitive Response In Tobacco Research Articles

First report of Pseudomonas cichorii causing bacterial leaf blight of pocketbook plant (Calceolaria hybrida) in Taiwan.

Pocketbook plants (Calceolaria spp.) are flowering ornamentals often grown as potted plants (Poesch 1937). In December 2022, leaf blight symptoms were observed on 2-mo-old plants of C. hybrida F1 'Dainty'. The disease was found in a nursery in Ren'ai Township, Nantou, and about 20% of the plants exhibited symptoms. Symptomatic plants had brown or gray necrotic lesions of different sizes and shapes, mostly around leaf margins. Lower leaf wilting was also observed (Fig. S1, A and B). Three plants were sampled. Leaf lesions were surface-disinfected with 75% ethanol and cut into smaller pieces in 10 mM MgCl2. After observing bacterial streaming under a microscope, the bacteria were streaked onto nutrient agar (NA). Following 2 days at 28°C, a type of round, creamy white colony predominated on all the plates. Three strains (Calc-A, Calc-B, and Calc-C) were obtained, one from each plant. The strains produced fluorescent pigments on King's B medium and were tested Gram-negative. The strains were characterized with the LOPAT scheme (Schaad et al. 2001). They did not exhibit activities of pectic enzymes, arginine dihydrolase and levan sucrase, but produced oxidase and induced the hypersensitive response in tobacco. DNA was extracted from the strains for PCR amplification of the 16S rDNA with primer pair 27f/1492r as described by Lane (1991). The 16S rDNA sequences were compared with entries in the GenBank database. The sequences obtained (GenBank accession no. OR824302) matched that of Pseudomonas cichorii MAFF 301158 (accession no. AB724288; 1,403/1,403 bp) and were 99% identical to that of DSM 50259T (accession no. CP074349; 1,391/1,405 bp). The strains were also tested with the species-specific primers hrp1a and hrp2a (Cottyn et al. 2011). The amplicons were sequenced and a BLASTn search showed that the sequences (accession no. OR827305) shared the highest identity (99.3%) with that of P. cichorii strain 83-1 (accession no. DQ168848; 848/854 bp) and were 97.3% identical to the sequence of DSM 50259T (accession no. CP074349; 831/854 bp). Calc-A was selected as a representative strain and deposited in the Bioresource Collection and Research Center, Taiwan (reference no. BCRC 81432). Koch's postulates were fulfilled by spray-inoculating a suspension of Calc-A on three 2-mo-old C. hybrida F1 'Dainty' plants. The inoculum was prepared by suspending NA-grown cells in 10 mM MgCl2 including 0.02% Silwet L-77 (OD600 = 0.3; 1.5 x 108 CFU/ml). For the controls, three plants were sprayed with bacteria-free solution. The plants were bagged throughout the experiment and kept in a growth chamber (14/10 h light/dark; 26/24°C day/night). Leaf blight and wilting symptoms developed on all leaves of the inoculated plants after 30 h, but not the controls (Fig. S1, C and D). The pathogen was reisolated from the treatment group, and colony PCR with hrp1a/hrp2a showed that the reisolated strain shared the same sequence with Calc-A to Calc-C. Repeating the inoculation assay produced consistent results. This is the first report of P. cichorii affecting Calceolaria in Taiwan. The bacterium has been reported infecting diverse crops in Taiwan, such as tomato and lettuce (Tsai et al. 2014). Expanding the understanding of the pathogen's potential hosts could help prevent its spread across important crops.

Identification of a hypersensitive response core peptide of HrpZ and its role in increasing grape downy mildew resistance

Harpins play a key role in inducing disease resistance in crops, and identifying their core functional regions and establishing a system for their efficient expression would be very valuable. In this study, large amounts of soluble fusion proteins of harpin HrpZ and its subpeptides were obtained via the optimized induction conditions (28 °C with 0.5 mmol·L−1 IPTG for 6 h) in Escherichia coli BL21 (DE3). Hypersensitive response (HR) assays demonstrated that the C-terminal 66 aa of HrpZ (HrpZ_C_2_2) elicited a strong HR in tobacco (Nicotiana benthamiana) and grape (Flame Seedless) leaves. Additionally, treatment with HrpZ, and particularly HrpZ_C_2_2, significantly reduced the disease incidence and severity index of field vine leaves and those inoculated with downy mildew. The determination of the physiological parameters indicated that HrpZ, and especially HrpZ_C_2_2, improved the photosynthesis- and chlorophyll fluorescence-related parameters, enhanced the activity of defense-related enzymes, including SOD, POD, CAT and PAL, and increased the H2O2 level. Collectively, we efficiently expressed a core peptide of HrpZ and elucidated its strong ability to elicit a HR and resistance to downy mildew. This research provides insight into understanding the structure and function of HrpZ and will advance the application of HrpZ_C_2_2 to increase the resistance of grapevine to downy mildew.

IN VITRO STUDY OF PHYLLOSPHERE BACTERIA AS PROMISING BIOCONTROL AGENTS AGAINST BACTERIAL LEAF BLIGHT DISEASE (Xanthomonas oryzae pv. oryzae) IN RICE PLANTS

This study aimed to explore the potential of phyllosphere bacterial isolates as biological control agents against Xanthomonas oryzae pv. oryzae (Xoo). We isolated a strain exhibiting typical morphological and biochemical characteristics of Xoo, such as a yellowish, spherical colony, Gram-negative, and starch hydrolyzing ability. This isolate was shown to cause necrotic lesions on rice leaves, corroborating its pathogenic nature. Subsequently, we isolated ten diverse phyllosphere bacteria from rice plants. These isolates were characterized based on various morphological and biochemical attributes including colony shape, margin appearance, color, growth time, and the ability to fluoresce under UV light when cultivated on King's B medium. Crucially, none of these isolates induced a hypersensitive response in tobacco leaves, an initial indicator of their potential as safe biological control agents. In an antagonistic assay against Xoo, all the phyllosphere isolates demonstrated varying levels of inhibition, suggesting their potential role in biological control. Two isolates, PGM1 and PGM4, exhibited the highest antagonistic effects against Xoo. These findings provide preliminary evidence for the potential use of these phyllosphere isolates in managing bacterial leaf blight in rice, warranting further research to confirm their efficacy and safety under field conditions.

First report of Robbsia andropogonis causing bacterial leaf spot of bougainvilleas in Taiwan.

Bougainvilleas (Bougainvillea spp.) are popular ornamentals commonly grown as bushes, vines, or trees worldwide (Kobayashi et al. 2007). Leaf spot symptoms were observed on a bougainvillea hedge located in North District, Taichung, Taiwan during August of 2022. The lesions were brown, necrotic and had yellow halos (Fig. S1). All the plants at the location showed similar symptoms. Leaf samples were collected from five plants and symptomatic tissues were minced in 10 mM MgCl2. The samples were streaked onto nutrient agar (NA) and after culturing at 28°C for 2 days, small, round, creamy white colonies were consistently isolated from all the samples. A total of five strains (BA1 to BA5) were obtained; each of them was isolated from a different plant. All five strains induced hypersensitive response in tobacco leaves. Amplification and sequencing of the isolated strains' 16S rDNA using primers 27F and 1492R (Lane 1991) revealed that all five strains shared identical sequences (GenBank accession no. OQ053015) with Robbsia andropogonis LMG 2129T (formerly Burkholderia andropogonis and Pseudomonas andropogonis; GenBank accession no. NR104960; 1,393/1,393 bp). Further testing of BA1 to BA5's DNA samples using the pathogen's species-specific primers Pf (5'-AAGTCGAACGGTAACAGGGA-3') and Pr (5'-AAAGGATATTAGCCCTCGCC-3'; Bagsic et al. 1995) successfully amplified the expected 410-bp amplicon in all five samples; the sequences of the PCR products completely matched to those of BA1 to BA5's 16S rDNA. Strains BA1 to BA5 also tested negative for arginine dihydrolase and oxidase activity, and failed to grow at 40°C, all of which are consistent with descriptions of R. andropogonis (Schaad et al. 2001). Pathogenicity of the isolated bacteria was confirmed by spray inoculation. Three representative strains, BA1 to BA3, were used for the assay. Bacterial colonies were scraped from NA plates and suspended in 10 mM MgCl2 added with 0.02% Silwet L-77. The concentrations of the suspensions were adjusted to 4.4-5.8 x 108 cfu/ml. The suspensions were sprayed onto three-month-old, cutting-propagated bougainvillea plants (to runoff). Controls were treated with bacteria-free solutions. Three plants were used for each treatment group (and the controls). The plants were placed in a growth chamber (27/25°C, day/night; 14-hour photoperiod) and bagged for three days. Within 20 days post inoculation, brown, necrotic lesions resembling those observed in the sampling site were observed on all inoculated plants, but not on the controls. One strain was re-isolated for each treatment group and the re-isolated strains all shared the same colony morphology and 16S rDNA sequence with BA1 to BA5. Additional PCR testing of these re-isolated strains using Pf and Pr also produced the expected amplicon. This is the first formal report of R. andropogonis affecting bougainvilleas in Taiwan. The pathogen has been reported causing diseases of betel palm (Areca catechu), corn and sorghum in Taiwan (Hseu et al. 2007; Hsu et al. 1991), some of which are economically important (Lisowicz 2000; Navi et al. 2002). As such, infected bougainvilleas could potentially serve as an inoculum source for these diseases.

First report of wildfire disease caused by Pseudomonas amygdali pv. tabaci on perilla in Korea

Perilla (Perilla frutescens) is an important crop which produces edible leaves and oil from its seeds which is used as a seasoning in Korean food (Kim et al., 2009). Perilla is cultivated throughout the year in greenhouses in southern Korea. In November 2020, irregular brown spots with halos that led to necrosis on most upper leaves were observed in Miryang (35°32´04.6˝N; 128°46´01.9˝E) (Figure 1-3). The symptoms were very similar to wildfire disease in soybeans. The disease spread to up to 20–30% of the perilla plants cultivated in two greenhouses. Five diseased plants were collected and the leaves of each plant were removed. To isolate the causal pathogen, supernatants of surface-sterilised and homogenised leaves were plated on King's B medium (Sigma-Aldrich, USA) and incubated at 28°C for two days. Fluorescent colonies occupied approximately 60% of the area covered by bacterial colonies, indicating that a fluorescent bacterium was the predominant isolate from the diseased perilla. One isolate (designated strain MY1) was collected for analyses of 16S rRNA gene sequences and multilocus sequence analysis (MLSA) using sequences of the gltA, gyrB, and rpoD genes (Hwang et al., 2005). A BLAST search showed that the 16S rRNA gene sequence of strain MY1 (GenBank Accession No. ON810536) corresponded to Pseudomonas amygdali strain DEB with 99% identity. Moreover, strain MY1 was closest to P. amygdali pv. tabaci strain ATCC 11528 in a phylogenetic tree constructed using MLSA (Yun & Kim, 2021) (Figure 4). The physiological and biochemical characteristics of strain MY1 were determined based on levan production, oxidase activity, potato soft rot, arginine dihydrolase activity and tobacco hypersensitive response (LOPAT) tests. In the tests, strain MY1 was positive for levan production and tobacco hypersensitivity, but negative for oxidase activity, potato soft rot and arginine dihydrolase activity. Based on the LOPAT and MLSA analyses, strain MY1 belongs to P. amygdali pv. tabaci 1a (Sun et al., 2021). To substantiate the pathogenicity of strain MY1, a bacterial suspension of the strain (adjusted to 108 CFU/ml) was inoculated onto ten four-week-old perilla and incubated at 28°C. After seven days, the inoculated perilla leaves showed similar symptoms to those observed originally in the greenhouse. Completing Koch's postulates, bacteria with fluorescent colonies were re-isolated from symptomatic tissue and the 16S rRNA sequence of the isolates was identical to P. amygdali. To the best of our knowledge, this is the first report of wildfire disease caused by P. amygdali pv. tabaci on perilla in Korea and globally. Perilla wildfire disease reduces the quality of leaves, so early detection and prevention are essential to reduce the spread of disease. S.Y. Choi and J.I. Kim contributed equally to this work. This work was supported by “Cooperative Research Program for Agriculture Science & Technology Development project (PJ01425901)” Rural Development Administration, Korea.

Type III secretion system genes hrcJ and hrpE affect virulence, hypersensitive response and biofilm formation of group II strains of Acidovorax citrulli

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli is a worldwide threat to watermelon and melon production. The type III secretion system (T3SS) plays an important role in the virulence of A. citrulli in its host plants and to induce hypersensitive response (HR) in its non-host plants. Little is known, however, about the contribution of the T3SS to biofilm formation in A. citrulli. We mutated two T3SS-related genes hrcJ and hrpE, respectively, and compared the mutants with their wild-type strain Aac-5 of A. citrulli, and their complementation strains on virulence, HR, and biofilm formation. Both mutants significantly reduced virulence in watermelon and melon seedlings and their ability to induce HR in tobacco leaves. Such reduction phenotypes were significantly recovered to the wild-type level, when the mutant strains were complemented with the wild-type hrcJ and hrpE genes. Interestingly, the two T3SS-related gene mutants also displayed enhanced ability to form biofilm, suggesting a different role of biofilm in the virulence of the group II stains of A. citrulli.

Evidence of Greater Competitive Fitness of Erwinia amylovora over E. pyrifoliae in Korean Isolates.

Erwinia amylovora and E. pyrifoliae are the causative agents of destructive diseases in both apple and pear trees viz. fire blight and black shoot blight, respectively. Since the introduction of fire blight in Korea in 2015, the occurrence of both pathogens has been independently reported. The co-incidence of these diseases is highly probable given the co-existence of their pathogenic bacteria in the same trees or orchards in a city/district. Hence, this study evaluated whether both diseases occurred in neighboring orchards and whether they occurred together in a single orchard. The competition and virulence of the two pathogens was compared using growth rates in vitro and in planta. Importantly, E amylovora showed significantly higher colony numbers than E. pyrifoliae when they were co-cultured in liquid media and co-inoculated into immature apple fruits and seedlings. In a comparison of the usage of major carbon sources, which are abundant in immature apple fruits and seedlings, E. amylovora also showed better growth rates than E. pyrifoliae. In virulence assays, including motility and a hypersensitive response (HR), E. amylovora demonstrated a larger diameter of travel from the inoculation site than E. pyrifoliae in both swarming and swimming motilities. E. amylovora elicited a HR in tobacco leaves when diluted from 1:1 to 1:16 but E. pyrifoliae does not elicit a HR when diluted at 1:16. Therefore, E. amylovora was concluded to have a greater competitive fitness than E. pyrifoliae.

Konya İli Böğürtlen, Ahududu ve Kuşburnu Yetiştiriciliğinde Potansiyel Bir Tehdit: Ateş Yanıklığı Hastalığı

In the present study, totally 49 samples, which showed the symptoms of leaf and shoot blight and cankers with brown discoloration of necrotic tissues on mature branches, were collected from 22 districts and areas of Konya Province between 2017 and 2019. Presence rate of E. amylovora in collected samples, showing symptoms of the disease, from the province was determined to be 40% for blackberry and raspberry and 33% rosehip for rosehip in three years. Bacteria consistently isolated from the diseased tissues were identified on the basis of biochemical, physiological, and molecular tests, comparing with a reference strain of E. amylovora, isolated from blackberry (Kbb 371). Twenty seven representative bacterial strains were gram-negative, rod-shaped, mucoid, fermentative, positive for levan formation and acetoin production, no growth at 36°C, positive for gelatin hydrolysis, and negative for esculin hydrolysis, indole, urease, catalase, oxidase, arginine dehydrolase, reduction of nitrate, acid production from lactose, and inositol. All strains induced a hypersensitive response in tobacco (Nicotiana tobacum cv. White Burley) 24 h after inoculation with a 108 CFU ml-1 bacterial suspension in sterile distilled water. The strains were identified as E. amylovora using the species-specific primers set A/B (1), which amplified a 1-kb DNA fragment in PCR, and the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method. In order to fulfill the Koch postulates, pathogenicity test was confirmed by injecting bacterial suspensions of 108 CFU ml-1 in sterile distilled water into the shoot tips of 3-year-old blackberry R. fruticosus cv. Chester, raspberry R. idaeus cv. Heritage and rosehip R. canina. All tests were repeated three times. The bacterium was re-isolated from inoculated plants and identified as E. amylovora. Phytosanitary measures are needed to prevent any further spread of the bacterium as potential inoculum sources to new blackberry, raspberry and rosehip growing areas.

Overexpression of PsoRPM3, an NBS-LRR gene isolated from myrobalan plum, confers resistance to Meloidogyne incognita in tobacco.

We reported an NBS-LRR gene, PsoRPM3, is highly expressed following RKN infection, initiating an HR response that promotes plant resistance. Meloidogyne spp. are root-knot nematodes (RKNs) that cause substantial economic losses worldwide. Screening for resistant tree resources and identifying plant resistance genes is currently the most effective way to prevent RKN infestations. Here, we cloned a novel TIR-NB-LRR-type resistance gene, PsoRPM3, from Xinjiang wild myrobalan plum (Prunus sogdiana Vassilcz.) and demonstrated that its protein product localized to the nucleus. In response to Meloidogyne incognita infection, PsoRPM3 gene expression levels were significantly higher in resistant myrobalan plum plants compared to susceptible plants. We investigated this difference, discovering that the - 309 to - 19bp region of the susceptible PsoRPM3 promoter was highly methylated. Indeed, heterologous expression of PsoRPM3 significantly enhanced the resistance of susceptible tobacco plants to M. incognita. Moreover, transient expression of PsoRPM3 induced a hypersensitive response in tobacco, whereas RNAi-mediated silencing of PsoRPM3 in transgenic tobacco reduced this hypersensitive response. Several hypersensitive response marker genes were considerably up-regulated in resistant myrobalan plum plants when compared with susceptible counterparts inoculated with M. incognita. PsoPR1a (a SA marker gene), PsoPR2 (a JA marker gene), and PsoACS6 (an ET signaling marker gene) were all more highly expressed in resistant than in susceptible plants. Together, these results support a model in which PsoRPM3 is highly expressed following RKN infection, initiating an HR response that promotes plant resistance through activated salicylic acid, jasmonic acid, and ethylene signaling pathways.

The stability of the coiled-coil structure near to N-terminus influence the heat resistance of harpin proteins from Xanthomonas

BackgroundHeat resistance is a common characteristic of harpins, a class of proteins found in Gram-negative bacteria, which may be related to the stability of coiled-coil (CC) structure. The CC structure is a ubiquitous protein folding and assembly motif made of α-helices wrapping around each other forming a supercoil. Specifically, whether the stability of the CC structure near to N-terminus of four selected harpin proteins from Xanthomonas (hereafter referred to as Hpa1) would influence their characteristics of heat resistance was investigated. We used bioinformatics approach to predict the structure of Hpa1, used the performance of hypersensitive response (HR)-induction activity of Hpa1 and circular dichroism (CD) spectral analyses to detect the relationship between the stability of the CC structure of Hpa1 and heat resistance.ResultsEach of four-selected Hpa1 has two α-helical regions with one in their N-terminus that could form CC structure, and the other in their C-terminus that could not. And the important amino acid residues involved in the CC motifs are located on helices present on the surface of these proteins, indicating they may engage in the formation of oligo mericaggregates, which may be responsible for HR elicitation by harpins and their high thermal stability. Increased or decreased the probability of forming a CC could either induce a stronger HR response or eliminate the ability to induce HR in tobacco after high temperature treatment. In addition, although the four Hpa1 mutants had little effect on the induction of HR by Hpa1, its thermal stability was significantly decreased. The α-helical content increased with increasing temperature, and the secondary structures of Hpa1 became almost entirely α-helices when the temperature reached 200 °C. Moreover, the stability of the CC structure near to N-terminus was found to be positively correlated with the heat resistance of Hpa1.ConclusionsThe stability of the CC structure might sever as an inner drive for mediating the heat resistance of harpin proteins. Our results offer a new insight into the interpretation of the mechanism involved in the heat resistance of harpin protein and provide a theoretical basis for further harpin function investigations and structure modifications.

Two Homologues of the Global Regulator Csr/Rsm Redundantly Control Phaseolotoxin Biosynthesis and Virulence in the Plant Pathogen Pseudomonas amygdali pv. phaseolicola 1448A.

The widely conserved Csr/Rsm (carbon storage regulator/repressor of stationary-phase metabolites) post-transcriptional regulatory system controls diverse phenotypes involved in bacterial pathogenicity and virulence. Here we show that Pseudomonas amygdali pv. phaseolicola 1448A contains seven rsm genes, four of which are chromosomal. In RNAseq analyses, only rsmE was thermoregulated, with increased expression at 18 °C, whereas the antagonistic sRNAs rsmX1, rsmX4, rsmX5 and rsmZ showed increased levels at 28 °C. Only double rsmA-rsmE mutants showed significantly altered phenotypes in functional analyses, being impaired for symptom elicitation in bean, including in planta growth, and for induction of the hypersensitive response in tobacco. Double mutants were also non-motile and were compromised for the utilization of different carbon sources. These phenotypes were accompanied by reduced mRNA levels of the type III secretion system regulatory genes hrpL and hrpA, and the flagellin gene, fliC. Biosynthesis of the phytotoxin phaseolotoxin by mutants in rsmA and rsmE was delayed, occurring only in older cultures, indicating that these rsm homologues act as inductors of toxin synthesis. Therefore, genes rsmA and rsmE act redundantly, although with a degree of specialization, to positively regulate diverse phenotypes involved in niche colonization. Additionally, our results suggest the existence of a regulatory molecule different from the Rsm proteins and dependent on the GacS/GacA (global activator of antibiotic and cyanide production) system, which causes the repression of phaseolotoxin biosynthesis at high temperatures.

Physiological and Transcriptional Response of Xanthomonas oryzae pv. oryzae to Berberine, an Emerging Chemical Control.

Berberine, a botanical drug, has great ability to inhibit the growth of Xanthomonas oryzae pv. oryzae. However, the antibacterial mechanism of berberine against X. oryzae pv. oryzae remains poorly understood. In this study, we investigated the physiological and transcriptional response of X. oryzae pv. oryzae to berberine. When strain X. oryzae pv. oryzae GX13 was treated with berberine (10 µg/ml), the hypersensitive response in tobacco, virulence to rice, pathogen population in the rice xylem, production of extracellular polysaccharide (EPS), and activity of extracellular hydrolases decreased, but the levels of pyruvate and ATP increased. Moreover, biofilm formation was inhibited, and the cell membrane was damaged. Transcriptome sequencing analysis showed downregulated expression of gspD, gspE, and gspF, involved in the type II secretion system (T2SS); hrcC, hrcJ, hrcN, and others, involved in the type III secretion system (T3SS); gumB and gumC, associated with EPS; zapE, ftsQ, and zapA, associated with cell division; lpxH, lpxK, kdtA, and others, associated with the membrane; and pyk, pgk, and mdh, encoding pyruvate kinase, phosphoglycerate kinase, and malate dehydrogenase, respectively. Upregulated expression was observed for nuoA, nuoB, and nuoH, encoding the NADH dehydrogenase complex, and atpF, atpC, and atpB, encoding ATP synthase. An adenylate cyclase (CyaA) fusion assay showed that berberine affects type three effector protein secretion via the T3SS and reduces effector translocation in X. oryzae pv. oryzae. It is speculated that the negative growth and virulence phenotypes of berberine-treated X. oryzae pv. oryzae GX13 may involve differentially expressed genes associated with cytoarchitecture and energy metabolism, and these effects on primary cell function may further dampen virulence and result in differential expression of T3SS- and T2SS-related genes.

A Novel Protein Elicitor PeBL2, from Brevibacillus laterosporus A60, Induces Systemic Resistance against Botrytis cinerea in Tobacco Plant.

Here, we reported a novel secreted protein elicitor PeBL2 from Brevibacillus laterosporus A60, which can induce hypersensitive response in tobacco (Nicotiana benthamiana). The ion-exchange chromatography, high-performance liquid chromatography (HPLC) and mass spectrometry were performed for identification of protein elicitor. The 471 bp PeBL2 gene produces a 17.22 kDa protein with 156 amino acids containing an 84-residue signal peptide. Consistent with endogenous protein, the recombinant protein expressed in Escherichia coli induced the typical hypersensitive response (HR) and necrosis in tobacco leaves. Additionally, PeBL2 also triggered early defensive response of generation of reactive oxygen species (H2O2 and O2–) and systemic resistance against of B. cinerea. Our findings shed new light on a novel strategy for biocontrol using B. laterosporus A60.

A novel protein elicitor PeFOC1 from Fusarium oxysporum triggers defense response and systemic resistance in tobacco

In recent years, it is a hotspot research field on interaction mechanism between elicitor and plant. In this study, a novel hypersensitive response (HR)-inducing protein elicitor was isolated from the culture filtrate of Fusarium oxysporum f. sp. cubense and named PeFOC1, which consisted of 321 amino acids with a molecular weight of approximately 35 kDa. After the inducible expression in Escherichia coli and purification by ÄKTA explore system, the recombinant PeFOC1 also triggered a typical HR in tobacco. In addition, PeFOC1 induced a cascade of defense response in tobacco including production of hydrogen peroxide, deposition of callose, and accumulation of phenolic compounds. Moreover, PeFOC1 significantly improved systemic resistance of tobacco seedlings to tobacco mosaic virus and Pseudomonas syringae pv. tabaci. Real-time quantitative-PCR analysis indicated that several defense-related genes in tobacco, such as NtPR1a, NtNPR1, NtPAL, NtEDS1, NtPDF, and NtLOX, were all up-regulated by the treatment of PeFOC1. All these results collectively demonstrated that PeFOC1 triggered defense response and systemic acquired resistance (SAR) in tobacco. This research not only provides further research on immune mechanism between plant and elicitor, but also sheds new light on strategy for biocontrol in the future.

Nitrite and nitric oxide are important in the adjustment of primary metabolism during the hypersensitive response in tobacco.

Nitrate and ammonia deferentially modulate primary metabolism during the hypersensitive response in tobacco. In this study, tobacco RNAi lines with low nitrite reductase (NiRr) levels were used to investigate the roles of nitrite and nitric oxide (NO) in this process. The lines accumulate NO2-, with increased NO generation, but allow sufficient reduction to NH4+ to maintain plant viability. For wild-type (WT) and NiRr plants grown with NO3-, inoculation with the non-host biotrophic pathogen Pseudomonas syringae pv. phaseolicola induced an accumulation of nitrite and NO, together with a hypersensitive response (HR) that resulted in decreased bacterial growth, increased electrolyte leakage, and enhanced pathogen resistance gene expression. These responses were greater with increases in NO or NO2- levels in NiRr plants than in the WT under NO3- nutrition. In contrast, WT and NiRr plants grown with NH4+ exhibited compromised resistance. A metabolomic analysis detected 141 metabolites whose abundance was differentially changed as a result of exposure to the pathogen and in response to accumulation of NO or NO2-. Of these, 13 were involved in primary metabolism and most were linked to amino acid and energy metabolism. HR-associated changes in metabolism that are often linked with primary nitrate assimilation may therefore be influenced by nitrite and NO production.

A Na+ /Ca2+ exchanger of the olive pathogen Pseudomonas savastanoi pv. savastanoi is critical for its virulence.

SummaryIn a number of compatible plant‐bacterium interactions, a rise in apoplastic Ca2+ levels is observed, suggesting that Ca2+ represents an important environmental clue, as reported for bacteria infecting mammalians. We demonstrate that Ca2+ entry in Pseudomonas savastanoi pv. savastanoi (Psav) strain DAPP‐PG 722 is mediated by a Na+/Ca2+ exchanger critical for virulence. Using the fluorescent Ca2+ probe Fura 2‐AM, we demonstrate that Ca2+ enters Psav cells foremost when they experience low levels of energy, a situation mimicking the apoplastic fluid. In fact, Ca2+ entry was suppressed in the presence of high concentrations of glucose, fructose, sucrose or adenosine triphosphate (ATP). Since Ca2+ entry was inhibited by nifedipine and LiCl, we conclude that the channel for Ca2+ entry is a Na+/Ca2+ exchanger. In silico analysis of the Psav DAPP‐PG 722 genome revealed the presence of a single gene coding for a Na+/Ca2+ exchanger (cneA), which is a widely conserved and ancestral gene within the P. syringae complex based on gene phylogeny. Mutation of cneA compromised not only Ca2+ entry, but also compromised the Hypersensitive response (HR) in tobacco leaves and blocked the ability to induce knots in olive stems. The expression of both pathogenicity (hrpL, hrpA and iaaM) and virulence (ptz) genes was reduced in this Psav‐cneA mutant. Complementation of the Psav‐cneA mutation restored both Ca2+ entry and pathogenicity in olive plants, but failed to restore the HR in tobacco leaves. In conclusion, Ca2+ entry acts as a ‘host signal’ that allows and promotes Psav pathogenicity on olive plants.

Integration Host Factor Is Essential for Biofilm Formation, Extracellular Enzyme, Zeamine Production, and Virulence in Dickeya zeae.

Dickeya zeae is a globally important pathogenic bacterium that infects many crops, including rice, maize, potato, and banana. Bacterial foot rot of rice caused by D. zeae is one of the most important bacterial diseases of rice in China and some Southeast Asian countries. To investigate the functions of integration host factor (IHF) in D. zeae, we generated knockout mutants of ihfA and ihfB. Phenotypic assays showed that both the ΔihfA and ΔihfB strains had greatly reduced mobility, biofilm formation, extracellular protease, and pectinase activities, and toxin production compared with the wild-type strain. In addition, the mutants did not inhibit the germination of rice seeds, failed to cause soft rot in potatoes and a hypersensitive response in tobacco, and were avirulent in rice. Quantitative reverse-transcription polymerase chain reaction analysis demonstrated that IHF positively regulates the expression of zmsA, hrpN/Y, pelA/B/C, pehX, celZ, prtG, fliC, and DGC (diguanylate cyclase). Electrophoretic mobility shift assays further confirmed that IhfA binds to the promoter region of the DGC gene and may alter the levels of a second bacterial messenger, c-di-GMP, to regulate the pathogenicity or other physiological functions of D. zeae. In summary, IHF is an important integrated regulator of pathogenicity in D. zeae.

Two virulent sRNAs identified by genomic sequencing target the type III secretion system in rice bacterial blight pathogen

BackgroundSmall non-coding RNA (sRNA) short sequences regulate various biological processes in all organisms, including bacteria that are animal or plant pathogens. Virulent or pathogenicity-associated sRNAs have been increasingly elucidated in animal pathogens but little is known about similar category of sRNAs in plant-pathogenic bacteria. This is particularly true regarding rice bacterial blight pathogen Xanthomonas oryzae pathovar oryzae (Xoo) as studies on the virulent role of Xoo sRNAs is very limited at present.ResultsThe number and genomic distribution of sRNAs in Xoo were determined by bioinformatics analysis based on high throughput sequencing (sRNA-Seq) of the bacterial cultures from virulence-inducing and standard growth media, respectively. A total of 601 sRNAs were identified in the Xoo genome and ten virulent sRNA candidates were screened out based on significant differences of their expression levels between the culture conditions. In addition, trans3287 and trans3288 were also selected as candidates due to high expression levels in both media. The differential expression of 12 sRNAs evidenced by the sRNA-Seq data was confirmed by a convincing quantitative method. Based on genetic analysis of Xoo ΔsRNA mutants generated by deletion of the 12 single sRNAs, trans217 and trans3287 were characterized as virulent sRNAs. They are essential not only for the formation of bacterial blight in a susceptible rice variety Nipponbare but also for the induction of hypersensitive response (HR) in nonhost plant tobacco. Xoo Δtrans217 and Δtrans3287 mutants fail to induce bacterial blight in Nipponbare and also fail to induce the HR in tobacco, whereas, genetic complementation restores both mutants to the wild type in the virulent performance and HR induction. Similar effects of gene knockout and complementation were found in the expression of hrpG and hrpX genes, which encode regulatory proteins of the type III secretion system. Consistently, secretion of a type III effector, PthXo1, is blocked in Δtrans217 or Δtrans3287 bacterial cultures but retrieved by genetic complementation to both mutants.ConclusionsThe genetic analysis characterizes trans217 and trans3287 as pathogenicity-associated sRNAs essential for the bacterial virulence on the susceptible rice variety and for the HR elicitation in the nonhost plant. The molecular evidence suggests that both virulent sRNAs regulate the bacterial virulence by targeting the type III secretion system.

The Maize NBS-LRR Gene ZmNBS25 Enhances Disease Resistance in Rice and Arabidopsis.

Nucleotide-binding site-leucine-rich repeat (NBS-LRR) domain proteins are immune sensors and play critical roles in plant disease resistance. In this study, we cloned and characterized a novel NBS-LRR gene ZmNBS25 in maize. We found that ZmNBS25 could response to pathogen inoculation and salicylic acid (SA) treatment in maize, and transient overexpression of ZmNBS25 induced a hypersensitive response in tobacco. High-performance liquid chromatography (HPLC) analysis showed that, compared to control plants, ZmNBS25 overexpression (ZmNBS25-OE) in Arabidopsis and rice resulted in higher SA levels. By triggering the expression of certain defense-responsive genes, ZmNBS25-OE enhanced the resistance of Arabidopsis and rice to Pseudomonas syringae pv. tomato DC3000 and sheath blight disease, respectively. Moreover, we found little change of grain size and 1000-grain weight between ZmNBS25-OE rice lines and controls. Together, our results suggest that ZmNBS25 can function as a disease resistance gene across different species, being a valuable candidate for engineering resistance in breeding programs.

Characterization of response regulator GacA involved in phaseolotoxin production, hypersensitive response and cellular processes in Pseudomonas syringae pv. actinidiae A18

Pseudomonas syringae pv. actinidiae is the phytopathogen responsible for kiwifruit canker, one of the most important kiwifruit bacterial diseases worldwide. In this work, two phaseolotoxin-related genes (gacA and glmS) were cloned and identified through transposon mutagenesis in conjunction with Escherichia coli toxin bioassay, which resulted in completely abolishment in the production of phaseolotoxin in P. syringae pv. actinidiae A18. Further study showed that motility ability, exopolysaccharides production and biofilm formation were reduced in the gacA-defective mutant. Moreover, a diminished hypersensitive response in tobacco was observed with the gacA-defective mutant when compared to wild-type strain after infiltration. Our experimental results represented a first view that GacA might play a key role in controlling phaseolotoxin biosynthesis, hypersensitive response and cellular processes in this bacterium.