A Rapid Method for Screening Pathogen-Associated Molecular Pattern-Triggered Immunity-Intensifying Microbes.

PAMP-triggered immunity (PTI) is the first layer of plant defense response that occurs on the plant plasma membrane. Recently, the application of a rhizobacterium, Bacillus amyloliquefaciens strain PMB05, has been demonstrated to enhance flg22Pst- or harpin-triggered PTI response such as callose deposition. This PTI intensification by PMB05 further contributes to plant disease resistance to different bacterial diseases. Under the demand for rapid and large-scale screening, it has become critical to establish a non-staining technology to identify microbial strains that can enhance PTI responses. Firstly, we confirmed that the expression of the GSL5 gene, which is required for callose synthesis, can be enhanced by PMB05 during PTI activation triggered by flg22 or PopW (a harpin from Ralstonia solanacearum). The promoter region of the GSL5 gene was further cloned and fused to the coding sequence of gfp. The constructed fragments were used to generate transgenic Arabidopsis plants through a plant transformation vector. The transgenic lines of AtGSL5-GFP were obtained. The analysis was performed by infiltrating flg22Pst or PopW in one homozygous line, and the results exhibited that the green fluorescent signals were observed until after 8 h. In addition, the PopW-induced fluorescent signal was significantly enhanced in the co-treatment with PMB05 at 4 h after inoculation. Furthermore, by using AtGSL5-GFP to analyze 13 Bacillus spp. strains, the regulation of PopW-induced fluorescent signal was observed. And, the regulation of these fluorescent signals was similar to that performed by callose staining. More importantly, the Bacillus strains that enhance PopW-induced fluorescent signals would be more effective in reducing the occurrence of bacterial wilt. Taken together, the technique by using AtGSL5-GFP would be a promising platform to screen plant immunity-intensifying microbes to control bacterial wilt.

Bacillus amyloliquefaciens Strain PMB05 Intensifies Plant Immune Responses to Confer Resistance Against Bacterial Wilt of Tomato.

Tomato is an economic crop worldwide. Many limiting factors reduce the production of tomato, with bacterial wilt caused by Ralstonia solanacearum being the most destructive disease. Our previous study showed that the disease resistance to bacterial soft rot is enhanced by Bacillus amyloliquefaciens strain PMB05. This enhanced resistance is associated with the intensification of pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI). To determine whether the PTI-intensifying Bacillus spp. strains are able to confer disease resistance to bacterial wilt, their effects on PTI signals triggered by PAMP from R. solanacearum and on the occurrence of bacterial wilt were assayed. Before assay, a gene that encodes harpin from R. solanacearum, PopW, was applied as a PAMP. Results revealed that the B. amyloliquefaciens strain PMB05 was the one strain among 9 Bacillus rhizobacterial strains which could significantly intensify the PopW-induced hypersensitive response (HR) on Arabidopsis leaves. Moreover, we observed that the signals of PopW-induced reactive oxygen species generation and callose deposition were increased, confirming that the PTI was intensified by PMB05. The intensification of the PopW-triggered HR by PMB05 in Arabidopsis was reduced upon treatment with inhibitors in PTI pathways. Furthermore, the application of Bacillus spp. strains on tomato plants showed that only the use of PMB05 resulted in significantly increased resistance to bacterial wilt. Moreover, the PTI signals were also intensified in the tomato leaves. Taken together, we demonstrated that PMB05 is a PTI-intensifying bacterium that confers resistance to tomato bacterial wilt. Screening of plant immunity intensifying rhizobacteria is a possible strategy to control tomato bacterial wilt.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Using Dynamic Changes of Chlorophyll Fluorescence in Arabidopsis thaliana to Evaluate Plant Immunity-Intensifying Bacillus spp. Strains.

The integral defense responses of plants triggered by the small molecules of plant pathogens are regarded as plant immunity. The pathogen-associated molecular pattern-triggered immunity (PTI) occurs on the recognition of a pathogen by receptors on plant cell surfaces as an infection begins. During the activation of PTI, the effectiveness of a plant's photosynthetic system may be altered. In this study, chlorophyll fluorescence was used to assay the dynamic changes of PTI. When we used flg22Pst as an elicitor, we found that the photosynthetic electron transport rate (ETR) of Arabidopsis thaliana Col-0 was significantly decreased at 2, 4, and 24 h on treatment with a PTI-intensifying protein, plant ferredoxin-like protein (PFLP). In addition, this reduction in the photosynthetic ETR was also carried out with a PTI-intensifying Bacillus amyloliquefaciens strain, PMB05, on the induction of flg22Pst. The disease resistance against bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) was still enhanced by PMB05. Interestingly, among the eight tested Bacillus species strains, the PTI triggered by HrpNPcc from P. carotovorum subsp. carotovorum exhibited an ETR that was significantly decreased by PMB05. Furthermore, this decrease was consistent with rapid H2O2 generation and callose deposition triggered by HrpNPcc and the disease resistance against bacterial soft rot. Taken together, such results led us to conclude that the assay based on the ETR established in this study can be used as a model for evaluating the effectiveness of plant immunity-intensifying microbes for controlling plant diseases.

PFLP-Intensified Disease Resistance Against Bacterial Soft Rot Through the MAPK Pathway in PAMP-Triggered Immunity.

Bacterial soft rot is a devastating disease affecting a variety of vegetable crops worldwide. One strategy for controlling this disease could be the ectopic expression of the plant ferredoxin-like protein (pflp) gene. PFLP was previously shown to intensify pathogen-associated molecular pattern-triggered immunity (PTI), an immune response triggered, for example, by the flagellin epitope flg22. To gain further insight into how PFLP intensifies PTI, flg22 was used as an elicitor in Arabidopsis thaliana. First, PFLP was confirmed to intensify the rapid generation of H2O2, callose deposition, and the hypersensitive response when coinfiltrated with flg22. This response correlated with increased expression of the FLG22-induced receptor kinase 1 gene, which is part of the mitogen-activated protein kinase (MAPK) pathway. Although the increased response to flg22 alone did not depend on the MAPK pathway genes MEKK1, MKK5, and MPK6, the protective effect of PFLP decreased when plants mutated in these genes were inoculated with Pectobacterium carotovorum subsp. carotovorum. Furthermore, expression of PR1 and PDF1.2 also increased upon treatment with flg22 in the presence of PFLP. Taken together, these results suggest that activation of the MAPK pathway contributes to the increased resistance to bacterial soft rot observed in plants treated with PFLP.