Draft genome sequence, disease-resistance genes, and phenotype of a Paenibacillus terrae strain (NK3-4) with the potential to control plant diseases.

Paenibacillus terrae NK3-4 is a plant growth-promoting rhizobacterium that may be useful for controlling plant diseases. We conducted a genomic analysis and identified the genes mediating antimicrobial functions. Additionally, an extracellular antifungal protein component was isolated and identified. The draft genome sequence was assembled into 54 contigs, with 5 458 568 bp and a G+C content of 47%. Moreover, 4 690 015 bp encoded 5090 proteins, 7 rRNAs, and 54 tRNAs. Forty-four genes involved in antimicrobial functions were detected. They mainly encode 19 non-ribosomal peptide synthetases (NRPSs); one polyketide synthase/NRPSs hybrid enzyme; four Zn-dependent metalloproteases; three antilisterial bacteriocin subtilosin biosynthesis proteins (AlbA); four serine proteases; five pectate lyases; three beta-glucanases; and four 1,4-beta-xylanases. These include four novel NRPSs that have not been found in any species of Paenibacillus. Furthermore, five proteins exhibiting antifungal activity were identified from the antifungal extracellular protein component based on MS/MS and the strain NK3-4 predicted protein library. On the basis of these features, we propose that strain NK3-4 represents a promising biocontrol agent for protecting plant from diseases. The draft genome sequence described herein may provide the genetic basis for the characterization of the molecular mechanisms underlying the biocontrol functions. It may also facilitate the development of rational strategies for improving the strain.

Cloning and expression of the EsxA gene and the growth-promoting effects of the encoded protein on rice seedlings.

An EsxA-encoding gene (esxA) was previously identified in the genome of the plant growth-promoting rhizobacterium Paenibacillus terrae strain NK3-4. The esxA was cloned and expressed in Pichia pastoris, after which the effects of the EsxA protein on rice seedling growth were analyzed to determine whether EsxA contributes to the plant growth-promoting activity of strain NK3-4. The esxA was successfully cloned from the NK3-4 genome and ligated to the eukaryotic expression vector pPICZαA. The resulting pPICZαA-esxA recombinant plasmid was transinfected into yeast cells, and esxA expression in the yeast cells was confirmed. The treatment of seed- buds with the EsxA protein increased the root length by 1.35-times, but decreased the bud length. Additionally, in rice seedlings treated with EsxA, the root and shoot lengths increased by 2.6- and 1.7-times, respectively. These findings imply that EsxA is important for the promotion of rice plant growth by P. terrae strain NK3-4. Furthermore, the construction of the esxA expression vector and the engineered strain may be useful for future investigations of the mechanism underlying the plant growth-promoting effects of EsxA, with implications for the application of EsxA for regulating plant growth.

Protein Elicitor EsxA Induces Resistance to Seedling Blight and PR Genes Differential Transcription in Rice.

Protein elicitors can induce plant systemic resistance to pathogens. In an earlier study, we cloned an EsxA gene from the plant growth-promoting rhizobacterium Paenibacillus terrae NK3-4 and expressed it in Pichia pastoris. In addition to being important for the pathogenicity of animal pathogens, EsxA can also induce an immune response in animals. While, we found the exogenously expressed EsxA has the activity of elicitor, which can trigger hypersensitive response and reactive oxygen species burst in leaves as well as enhanced rice plant growth. The effects of EsxA on seedling blight (Fusarium oxysporum) resistance and gene transcription, including pathogenesis-related (PR) genes in rice were evaluated. The germination rate was 95.0% for seeds treated with EsxA and then inoculated with F. oxysporum, which was 2.8-times higher than that of F. oxysporum-infected control seeds that were not treated with EsxA (Con). The buds and roots of EsxA-treated seedlings were 2.4- and 15.9-times longer than those of Con seedlings. The plants and roots of seedlings dipped in an EsxA solution and then inoculated with F. oxysporum were longer than those of the Con seedlings. Theplant length, number of total roots, and number of white roots were respectively 23.2%, 1.74-times, and 7.42-times greater for the seedlings sprayed with EsxA and then inoculated with F. oxysporum than for the Con seedlings. The EsxA induction efficiency (spray treatment) on seedling blight resistance was 60.9%. The transcriptome analysis revealed 1137 and 239 rice genes with EsxA-induced up-regulated and down-regulated transcription levels, respectively. At 48 h after the EsxA treatment, the transcription of 611 and 160 genes was up-regulated and down-regulated, respectively, compared with the transcription levels for the untreated control at the same time-point. Many disease resistance-related PR genes had up-regulated transcription levels. The qPCR data were consistent with the transcriptome sequencing results. EsxA triggered rice ISR to seedling blight and gene differential transcription, including the up-regulated transcription of rice PR genes. These findings may be relevant for the use of EsxA as a protein elicitor to control plant diseases.