Interactive effects of Pseudomonas entomophila strain 23S and Clavibacter michiganensis subsp. michiganensis on proteome and anti-Cmm compound production.

Pseudomonas entomophila strain 23S is an effective biocontrol bacterium for tomato bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (Cmm); it produces an inhibitory compound affecting the growth of Cmm. In this study, the interactions between pure cultures of P. entomophila 23S and Cmm were investigated. First, the population dynamics of each bacterium during the interaction was determined using the selective media. Second, the amount of anti-Cmm compound produced by P. entomophila 23S in the presence of Cmm was quantified using HPLC. Lastly, a label-free shotgun proteomics study of P. entomophila 23S, Cmm, and a co-culture was conducted to understand the effects of the interaction of each bacterium at the proteomic level. Compared with the pure culture grown, the total number of proteins decreased in the interaction for both bacteria. P. entomophila 23S secreted stress-related proteins, such as chaperonins, peptidases, ABC-transporters and elongation factors. The bacterium also produced more proteins related with purine, pyrimidine, carbon and nitrogen metabolisms in the presence of Cmm. The population enumeration study revealed that the Cmm population declined dramatically during the interaction, while the population of P. entomophila 23S maintained. The quantification of anti-Cmm compound indicated that P. entomophila 23S produced significantly higher amount of anti-Cmm compound when it was cultured with Cmm. Overall, the study suggested that P. entomophila 23S, although is cidal to Cmm, was also negatively affected by the presence of Cmm, while trying to adapt to the stress condition, and that such an environment favored increased production of the anti-Cmm compound by P. entomophila 23S. SIGNIFICANCE: Pseudomonas entomophila strain 23S is an effective biocontrol bacterium for tomato bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (Cmm); it produces an inhibitory compound affecting the growth of Cmm. In this study, secreted proteome of pure cultures of P. entomophila 23S and Cmm, and also of a co-culture was first time identified. Furthermore, the study found that P. entomophila strain 23S produced significantly higher amount of anti-Cmm compound when the bacterium was grown together with Cmm. Co-culture enhancing anti-Cmm compound production by P. entomophila 23S is useful information, particularly from a commercial point of view of biocontrol application, and for scale-up of anti-Cmm compound production.

Lipo-chitooligosaccharide and thuricin 17 act as plant growth promoters and alleviate drought stress in Arabidopsis thaliana.

Lipo-chito-oligosaccharide (LCO-from Bradyrhizobium japonicum) and thuricin 17 (Th17-from Bacillus thuringiensis) are bacterial signal compounds from the rhizosphere of soybean that have been shown to enhance plant growth in a range of legumes and non-legumes. In this study, an attempt to quantify phytohormones involved in the initial hours after exposure of Arabidopsis thaliana to these compounds was conducted using UPLC-ESI-MS/MS. A petri-plate assay was conducted to screen for drought stress tolerance to PEG 8000 infusion and plant growth was studied 21-days post-stress. Arabidopsis thaliana plants grown in trays with drought stress imposed by water withhold were used for free proline determination, elemental analysis, and untargeted proteomics using LC-MS/MS studies. At 24 h post-exposure to the signal compounds under optimal growth conditions, Arabidopsis thaliana rosettes varied in their responses to the two signals. While LCO-treated rosettes showed a decrease in total IAA, cytokinins, gibberellins, and jasmonic acid, increases in ABA and SA was very clear. Th17-treated rosettes, on the other hand, showed an increase in IAA and SA. Both treatments resulted in decreased JA levels. Under severe drought stress imposed by PEG 8000 infusion, LCO and Th17 treatments were found to significantly increase fresh and dry weight over drought-stressed control plates, indicating that the presence of the signaling compounds decreased the negative effects experienced by the plants. Free proline content increased in LCO- and Th17-treated plants after water-withhold drought stress. Elemental analysis showed a significant increase in carbon percentage at the lower concentration of Th17. Untargeted proteomics revealed changes in the levels of drought-specific ribosomal proteins, glutathione S-transferase, late embryogenesis proteins, vegetative storage proteins 1 and 2, thaumatin-like proteins, and those related to chloroplast and carbon metabolism. The roles of some of these significantly affected proteins detected under drought stress are discussed.

Proteomic Studies on the Effects of Lipo-Chitooligosaccharide and Thuricin 17 under Unstressed and Salt Stressed Conditions in Arabidopsis thaliana.

Plants, being sessile organisms, are exposed to widely varying environmental conditions throughout their life cycle. Compatible plant-microbe interactions favor plant growth and development, and help plants deal with these environmental challenges. Microorganisms produce a diverse range of elicitor molecules to establish symbiotic relationships with the plants they associate with, in a given ecological niche. Lipo-chitooligosaccharide (LCO) and Thuricin 17 (Th17) are two such compounds shown to positively influence plant growth of both legumes and non-legumes. Arabidopsis thaliana responded positively to treatment with the bacterial signal compounds LCO and Th17 in the presence of salt stress (up to 250 mM NaCl). Shotgun proteomics of unstressed and 250 mM NaCl stressed A. thaliana rosettes (7 days post stress) in combination with the LCO and Th17 revealed many known, putative, hypothetical, and unknown proteins. Overall, carbon and energy metabolic pathways were affected under both unstressed and salt stressed conditions when treated with these signals. PEP carboxylase, Rubisco-oxygenase large subunit, pyruvate kinase, and proteins of photosystems I and II were some of the noteworthy proteins enhanced by the signals, along with other stress related proteins. These findings suggest that the proteome of A. thaliana rosettes is altered by the bacterial signals tested, and more so under salt stress, thereby imparting a positive effect on plant growth under high salt stress. The roles of the identified proteins are discussed here in relation to salt stress adaptation, which, when translated to field grown crops can be a crucial component and of significant importance in agriculture and global food production. The mass spectrometry proteomics data have been deposited to the ProteomeXchange with identifier PXD004742.

A micromolar concentration of lipo-chitooligosaccharide (Nod Bj V [C18:1, MeFuc]) regulates the emergence and seed productivity of rapid cycling canola (Brassica napus [L.]) plants.

The objective of this experiment was to assess whether or not the application of lipo-chitooligosaccharide (Nod Bj V [C18:1, MeFuc]) (LCO) would increase yield factors under conditions that would inhibit canola (Brassica napus L.) productivity. The seed application reduced the percentage of plants that were unproductive by 15.10% compared to plants grown from untreated seeds. Based on the 95% confidence interval for the difference, untreated plants would produce 38 to 3% fewer seeds than plants grown from LCO treated seeds. The experimental conditions were artificial, but further experimentation, with agricultural cultivars grown in greenhouses where natural conditions were simulated, confirmed that LCO treatment can contribute to canola yield.

A Proteomic Approach to Lipo-Chitooligosaccharide and Thuricin 17 Effects on Soybean GerminationUnstressed and Salt Stress.

Salt stress is an important abiotic stressor affecting crop growth and productivity. Of the 20 percent of the terrestrial earth's surface available as agricultural land, 50 percent is estimated by the United Nations Environment Program to be salinized to the level that crops growing on it will be salt-stressed. Increased soil salinity has profound effects on seed germination and germinating seedlings as they are frequently confronted with much higher salinities than vigorously growing plants, because germination usually occurs in surface soils, the site of greatest soluble salt accumulation. The growth of soybean exposed to 40 mM NaCl is negatively affected, while an exposure to 80 mM NaCl is often lethal. When treated with the bacterial signal compounds lipo-chitooligosaccharide (LCO) and thuricin 17 (Th17), soybean seeds (variety Absolute RR) responded positively at salt stress of up to 150 mM NaCl. Shotgun proteomics of unstressed and 100 mM NaCl stressed seeds (48 h) in combination with the LCO and Th17 revealed many known, predicted, hypothetical and unknown proteins. In all, carbon, nitrogen and energy metabolic pathways were affected under both unstressed and salt stressed conditions when treated with signals. PEP carboxylase, Rubisco oxygenase large subunit, pyruvate kinase, and isocitrate lyase were some of the noteworthy proteins enhanced by the signals, along with antioxidant glutathione-S-transferase and other stress related proteins. These findings suggest that the germinating seeds alter their proteome based on bacterial signals and on stress, the specificity of this response plays a crucial role in organ maturation and transition from one stage to another in the plants' life cycle; understanding this response is of fundamental importance in agriculture and, as a result, global food security. The mass spectrometry proteomics data have been deposited to the ProteomeXchange with identifier PXD004106.

Effects of Pseudomonas aureofaciens 63-28 on defense responses in soybean plants infected by Rhizoctonia solani.

The objective of this work was to investigate the ability of the plant growth-promoting rhizobacterium Pseudomonas aureofaciens 63-28 to induce plant defense systems, including defense-related enzyme levels and expression of defense-related isoenzymes, and isoflavone production, leading to improved resistance to the phytopathogen Rhizoctonia solani AG-4 in soybean seedlings. Seven-dayold soybean seedlings were inoculated with P. aureofaciens 63-28, R. solani AG-4, or P. aureofaciens 63-28 plus R. solani AG-4 (P+R), or not inoculated (control). After 7 days of incubation, roots treated with R. solani AG-4 had obvious damping-off symptoms, but P+R-treated soybean plants had less disease development, indicating suppression of R. solani AG-4 in soybean seedlings. Superoxide dismutase (SOD) and catalase (CAT) activities of R. solani AG-4-treated roots increased by 24.6% and 54.0%, respectively, compared with control roots. Ascorbate peroxidase (APX) and phenylalanine ammonia lyase (PAL) activities of R. solani AG-4-treated roots were increased by 75.1% and 23.6%, respectively. Polyphenol oxidase (PPO) activity in soybean roots challenged with P. aureofaciens 63-28 and P+R increased by 25.0% and 11.6%, respectively. Mn-SOD (S1 band on gel) and Fe-SOD (S2) were strongly induced in P+R-treated roots, whereas one CAT (C1) and one APX (A3) were strongly induced in R. solani AG-4- treated roots. The total isoflavone concentration in P+Rtreated shoots was 27.2% greater than the control treatment. The isoflavone yield of R. solani AG-4-treated shoots was 60.9% less than the control.

Induction of defense-related enzymes in soybean leaves by class IId bacteriocins (thuricin 17 and bacthuricin F4) purified from Bacillus strains.

We have recently discovered a new class of bacteriocin (class IId) which stimulates plant growth in a way similar to Nod factors. Nod factors have been shown to provoke aspects of plant disease resistance. We investigated the effects of bacteriocins [thuricin 17 (T17) and bacthuricin F4 (BF4)] on the activities of phenylalanine ammonia lyase (PAL), guaiacol peroxidase (POD), ascorbate peroxidase (APX), superoxide dismutase (SOD), and polyphenol oxidase (PPO). Bacteriocin solutions were fed into the cut stems of soybean (Glycine max L. Merr. cv. OAC Bayfield) seedlings at the first trifoliate stage. PAL activity in T17 treated leaves was the highest at 72h after treatment and was 75.5% greater than the control at that time. At 72h after treatment POD activities in T17 and BF4 treated leaves increased by 72.7 and 91.3%, respectively, as compared with the control treatment. APX activity was 52.3 and 49.6% respectively, greater than the control in T17 and BF4 treated leaves at 72h after treatment. SOD activity in T17 treated leaves was the highest at 72h after treatment and was 26.0% greater than the control at that time. SOD activity was 70.5 and 60.2% greater, respectively, than the control in T17 and BF4 treated leaves, at 72h. Using PAGE we found that one APX isozyme (28kDa isoform) showed the strongest induction in all bacteriocin treated leaves at 72h. Activity of the seven SOD isozymes was increased by both bacteriocins, relative to the control treatment. The 33kDa PPO isozyme was induced strongly by both bacteriocins, relative to the control treatment. These results indicate that class IId bacteriocins can act as an inducer of plant disease defense-related enzymes and may be acting through mechanisms similar to Nod factors.

The class IId bacteriocin thuricin-17 increases plant growth.

The mechanisms by which many plant growth promoting rhizobacteria (PGPR) affect plants are unknown. We recently isolated a rhizosphere bacterium (Bacillus thuringiensis NEB17), that promotes soybean growth and screened the liquid growth medium in which it grew for plant growth stimulating materials. We have also shown that it produces a bacteriocin (named by us as thuricin-17 and a member of the recently described class IId bacteriocins). Here we show that application of this bacteriocin to leaves (spray) or roots (drench) directly stimulates the growth of both a C(3) dicot (soybean) and a C(4) monocot (corn). This growth stimulation is similar in nature to that previously seen when plants are treated with Nod factors. Strain NEB17 contains three copies of the gene for thuricin 17 that code for identical amino acid sequences. These two lines of evidence suggest that the dual functions of these proteins may have constrained their evolution. This is the first report of direct plant growth enhancement by a bacteriocin.

Stability and antibacterial activity of bacteriocins produced by Bacillus thuringiensis and Bacillus thuringiensis ssp. kurstaki.

Bacteriocins are antimicrobial peptides that are produced by bacteria and toxic to bacterial strains closely related to the producer strain. It has previously been reported that Bacillus thuringiensis strain NEB17 and Bacillus thuringiensis subsp. kurstaki BUPM4 produce the bacteriocins thuricin 17 (3,162 Da) and bacthuricin F4 (3,160.05 Da), respectively. Here, we demonstrate that these bacteriocins have functional similarities and show a similar spectrum of antimicrobial activities against indicator strains. We also studied the effects of sterilization methods on the recovery and biological activities of these bacteriocins. They were completely degraded by autoclaving and the two were similarly affected by the tested filter membranes. Polyvinylidene fluoride (PVDF), polyestersulfone (PES), and cellulose acetate (CA) are suitable for filter sterilization of these bacteriocins. The two bacteriocins were stable across a range of storage conditions. These data will facilitate their utilization in food preservation or agricultural applications.

Chitinases produced by Paenibacillus illinoisensis and Bacillus thuringiensis subsp. pakistani degrade Nod factor from Bradyrhizobium japonicum.

Chitinases are enzymes that hydrolyze internal beta-1,4-N-acetyl-D-glucosamine linkages of chitin. Since the backbone of Nod factors is a chitin oligomer, we investigated whether chitinases produced by soil bacteria Paenibacillus illinoisensis KJA-424 and Bacillus thuringiensis subsp. Pakistani HD 395 are able to degrade Nod factor produced by Bradyrhizobium japonicum, a phenomenon that could disrupt B. japonicum-soybean signaling and nodule establishment when chitinases are present. Purified Nod factor [LCO Nod Bj-V (C(18:1), MeFuc)] was isolated from Bradyrhizobium japonicum and incubated with crude chitinases isolated from KJA-424 and HD395, with or without acetate buffer. After 15 h of incubation, Nod factor in the resulting solution was quantified by HPLC. Degradation was greatest following treatment with KJA-424 (91.9%) and HD395 (86.5%) chitinases in acetate buffer. Treatments that included acetate buffer had higher levels of degradation than those without. For all treatments degradation was greater than 77%.

Gas exchange characteristics and dry matter accumulation of soybean treated with Nod factors.

The effect of Nod factors on gas exchange characteristics and growth of soybean plants was studied. Soybean responded positively to some concentrations of Nod factors. Photosynthesis was increased up to 13% over the control, and this was accompanied by increases in stomatal conductance; plant dry weight was also increased. Near-fully expanded leaves responded more strongly to Nod factors than fully expanded leaves. Only Nod factor NodBj-V (C18:1, MeFuc) had significant effects on soybean. The finding suggests that Nod factors can affect photosynthesis, possibly indirectly, by stimulating sink strength.

Proteomic analysis of the bacteriocin thuricin 17 produced by Bacillus thuringiensis NEB17.

Thuricin 17 is a recently discovered bacteriocin produced by Bacillus thuringiensis NEB17. The objective of this work was to conduct a proteomic analysis of this bacteriocin. The partial N- and C-terminal amino-acid sequences of thuricin 17 have now been determined using the Edman degradation and matrix-assisted laser desorption ionization-quadrapole time of flight mass spectrometry (MS)/MS. A hydrophobic cluster analysis indicates that thuricin 17 contains a hydrophobic region, potentially corresponding to a membrane associated domain. Based on time of production, this bacteriocin may be produced as a secondary metabolite. Interestingly, thuricin 17 shares the same N-terminal sequence, DWTXWSXL, with a previously reported bacteriocin, Bacthuricin F4, produced by B. thuringiensis ssp. kurstaki strain BUPM4. This is the first time two bacteriocins from different Bacillus species have been shown to share similar N-terminal sequences.

Nod Bj-V (C18:1, MeFuc) production by Bradyrhizobium japonicum (USDA110, 532C) at suboptimal growth temperatures.

Nod factors (Lipo-chitooligosaccharides, or LCOs) act as bacteria-to-plant signal molecules that modulate early events of the Bradyrhizobium-soybean symbiosis. It is known that low root zone temperature inhibits the early stages of this symbiosis; however, the effect of low soil temperature on bacteria-to-plant signaling is largely uninvestigated. We evaluated the effect of low growth temperatures on the production kinetics of Nod factor (LCO) by B. japonicum. Two strains of B. japonicum, 532C and USDA110, were tested for ability to synthesize Nod Bj-V (C(18:1), MeFuc) at three growth temperatures (15, 17 and 28 degrees C). The greatest amounts of the major Nod factor, Nod Bj-V (C(18:1), MeFuc), were produced at 28 degrees C for both strains. At 17 and 15 degrees C, the Nod factor production efficiency, per cell, of B. japonicum 532C and USDA110 was markedly decreased with the lowest Nod factor concentration per cell occurring at 15 degrees C. Strain 532C was more efficient at Nod factor production per cell than strain USDA 110 at all growth temperatures. The biological activity of the extracted Nod factor was unaffected by culture temperature. This study constitutes the first demonstration of reduced Nod factor production efficiency (per cell production) under reduced temperatures, suggesting another way that lower temperatures inhibit establishment of the soybean N(2) fixing symbiosis.

Nod factor induces soybean resistance to powdery mildew.

Plants possess highly sensitive perception systems by which microbial signal molecules are recognized. In the Bradyrhizobium-soybean (Glycine max (L.) Merr.) symbiosis, recognition is initiated through exchange of signal molecules, generally flavonoids from soybean and lipo-chitooligosaccharides (Nod factors) from the microsymbiont. Application of the Nod factor Nod Bj-V (C18:1, MeFuc) induced soybean resistance to powdery mildew caused by Microsphaera diffusa. Addition of Nod factor (concentrations ranging from 10(-6) to 10(-10) M) to soybean root systems led to reductions in disease incidence. The lowest disease incidence was caused by Nod factor treatment at 10(-6) M. The effect of Nod factor application on fungal growth and development was measured at 4, 12, 48, and 96 h after inoculation. Colony diameter and number of germ tubes per conidium were decreased by 10(-6) M Nod factor. Phenylalanine ammonia lyase (PAL, EC.4.3.1.1.) is the first enzyme of the phenyl propanoid pathway, and is commonly activated as part of plant responses to disease. Treatment of soybean seedlings with Nod factor, through stem wounds, induced PAL activity; the most rapid increase followed treatment with 10(-6) M Nod factor. These data show that soybean plants are able to detect root applied LCO and respond by increased disease resistance.

In vitro induction of lipo-chitooligosaccharide production in Bradyrhizobium japonicum cultures by root extracts from non-leguminous plants.

Bradyrhizobium japonicum can form a N2-fixing symbiosis with compatible leguminous plants. It can also act as a plant-growth promoting rhizobacterium (PGPR) for non-legume plants, possibly through production of lipo-chitooligosaccharides (LCOs), which should have the ability to induce disease resistance responses in plants. The objective of this work was to determine whether non-leguminous crop plants can induce LCO formation by B. japonicum cultures. Cultures treated with root extracts of soybean, corn, cotton or winter wheat were assayed for presence and level of LCO. Root extracts of soybean, corn and winter wheat all induced LCO production, with extracts of corn inducing the greatest amounts. Root washings of corn also induced LCO production, but less than the root extract. These results indicated that the stimulation of non-legume plant growth by B. japonicum could be through the production of LCOs, induced by materials excreted by the roots of non-legume plants.

An inducible activator produced by a Serratia proteamaculans strain and its soybean growth-promoting activity under greenhouse conditions.

Serratia proteamaculans 1-102 (1-102) promotes soybean-bradyrhizobia nodulation and growth, but the mechanism is unknown. After adding isoflavonoid inducers to 1-102 culture, an active peak with a retention time of about 105 min in the HPLC fractionation was isolated using a bioassay based on the stimulation of soybean seed germination. The plant growth-promoting activity of this material was compared with 1-102 culture (cells) and supernatant under greenhouse conditions. The activator was applied to roots in 83, 830 and 8300 HPLC microvolts (microV) per seedling when plants were inoculated with bradyrhizobia or sprayed onto the leaves in same concentrations at 20 d after inoculation. The root-applied activator, especially at 1 ml of 830 microV per seedling, enhanced soybean nodulation and growth at the same level as 1-102 culture under both optimal and sub-optimal root zone temperatures. Thus, this activator stimulating soybean seed germination is also responsible for the plant growth-promoting activity of 1-102 culture. However, when sprayed onto the leaves, the activator did not increase growth and in higher concentrations decreased average single leaf area. The results suggest that this inducible activator might be a lipo-chitooligosaccharide (LCO) analogue. LCOs act as rhizobia-to-legume signals stimulating root nodule formation. The activator could provide additional 'signal', increasing in the signal quality (the signal-to-noise ratio, SNR) of the plant-rhizobia signal exchange process.