Lysin Motif (LysM) Proteins: Interlinking Manipulation of Plant Immunity and Fungi.

The proteins with lysin motif (LysM) are carbohydrate-binding protein modules that play a critical role in the host-pathogen interactions. The plant LysM proteins mostly function as pattern recognition receptors (PRRs) that sense chitin to induce the plant's immunity. In contrast, fungal LysM blocks chitin sensing or signaling to inhibit chitin-induced host immunity. In this review, we provide historical perspectives on plant and fungal LysMs to demonstrate how these proteins are involved in the regulation of plant's immune response by microbes. Plants employ LysM proteins to recognize fungal chitins that are then degraded by plant chitinases to induce immunity. In contrast, fungal pathogens recruit LysM proteins to protect their cell wall from hydrolysis by plant chitinase to prevent activation of chitin-induced immunity. Uncovering this coevolutionary arms race in which LysM plays a pivotal role in manipulating facilitates a greater understanding of the mechanisms governing plant-fungus interactions.

Psc-AFP from Psoralea corylifolia L. overexpressed in Pichia pastoris increases antimicrobial activity and enhances disease resistance of transgenic tobacco.

Psc-AFP, isolated from the seeds of Psoralea corylifolia L., is an antimicrobial protein with trypsin inhibitor activity. Its encoding gene was cloned by 3'- rapid amplification of cDNA ends (RACE) combined with Y-shaped adaptor-dependent extension (YADE) method. The gene Psc-AFP encodes a protein of 203 amino acids with a deduced signal peptide of 24 residues. The growth inhibition effect exerted by the heterologously expressed Psc-AFP in Pichia pastoris revealed that the recombinant Psc-AFP inhibited mycelium growth of Aspergillus niger, Rhizoctonia solani, and Alternaria brassicae and conidial germination of Alternaria alternata. The recombinant Psc-AFP also showed protease inhibitor activity manifested by the inhibition of trypsin. The transgenic tobacco bioassays confirmed that overexpressing Psc-AFP significantly enhanced the disease resistance of tobacco and that some of the transgenic lines were almost fully tolerant to Ralstonia solanacearum and A. alternata, whereas no apparent alteration in plant growth and development was observed. Collectively, these results indicate that the recombinant Psc-AFP is an active antimicrobial protein, with protease inhibitor activity that can be successfully produced in the yeast and tobacco and, therefore, maybe a potential antimicrobial candidate for practical use.

[Diversity of the Microbial Community in Rice Paddy Soil with Biogas Slurry Irrigation Analyzed by Illumina Sequencing Technology].

In order to explore the variation in soil microbial community diversity in paddy fields with different irrigation periods, we collected in situ rice field soils during different biogas irrigation periods and analyzed the microbial community structures of these soils by high-throughput sequencing. The results showed that as the biogas irrigation period increased, the soil pH decreased gradually, while organic matter, nitrate nitrogen, phosphate, and other nutrients were accumulated. Years of continued biogas irrigation was not conducive to improving rice yields. The results showed that as the biogas irrigation period increased, the richness in microbial species in paddy soils decreased gradually, and the diversity in the microbial communities was also reduced. Proteobacteria accounts for the largest proportion in rice paddy soil with biogas slurry irrigation. With the increase of biogas irrigation years, the proportion of ß-Proteobacteria, Bacteroidia, Bacteroidales, Burkholderiales, Bacteroides, and Thiobacillus increased, while the proportion of Gemmatimonadetes and α-Proteobacteria decreased gradually. Dissolved organic carbon (F=2.67, P=0.09) had the greatest effect on microbial community structures in the studied paddy soils.

[Cloning and characterization of cuticle degrading enzyme CDEP-1 from Beauveria bassiana].

Beauveria bassiana extracellular subtilisin-like serine endoprotease is a potential virulence factor by virtue of its activity against insect cuticles. A cDNA library was constructed using mRNA from mycelia of Beauveria bassiana grown on cuticle/chitin cultures. A cDNA clone of the protease, designated CDEP-1, was isolated from cDNA library. CDEP-1 contained an 1,134 bp ORF that predicted a protein of 377 amino acids with M(r) = 38,616 and PI = 8.302. The amino acid sequence of the gene shows 57.9%, 83.3% and 54.7% identity to Metarhizium nisopliae Pr1, Beauveria bassiana Pr1 and proteinase K, respectively. Southern analysis indicated that CDEP-1 was present as singly copy in Beauveria bassiana.

Isolation and characterization of a carboxylic transport protein JEN1 and its promoter from Metarhizium anisopliae.

Based on the flanking sequence of T-DNA of a T-DNA insertion mutant of Beauveria bassiana, T12, the full length cDNA of carboxylic transport protein, designated MaJen1, was cloned from Metarhizium anisopliae. MaJen1 is 1,695 bp long and contained a 1,524 bp ORF which predicted a protein of 508 amino acid. The amino acid sequence of the gene showed 69% and 31% identity to the carboxylic transport protein of Neurospore crassa and Saccharomyces cerevisiae, respectively. The genome sequence, GMaJen1, was amplified by PCR, indicating that there were two introns in GMaJen1. Southern analysis indicated that GMaJen1 was present as a singl copy in Metarhizium anisopliae. The result of RT-PCR showed that expression of MaJen1 was induced by the cuticle of cockroach and repressed by glucose. A 1,626 bp upstream sequence of GMaJen1 was amplified by YADE method, which contained several putative binding domains of glucose repressor.

[Cloning and characterization of a homologous gene of plant class V chitinase from balsampear, Momordica charantia Linn].

Balsampear (M. charantia Linn.) is a vegetable crop, highly resistant to pathogens. Chitinases were proposed to play an important role in the defense response of this crop. Based on the N-terminal sequence of a purified balsampear chitinase, a fragment (ChitB), similar to the tobacco class V chitinase gene, was amplified from the leaf RNA using 3'RACE, and the corresponding 5' sequence was further amplified by the Y-RACE method. By joining the two amplified fragments, the full-length cDNA of M. charatica homologous gene of plant class V chitinase (McChi5) was obtained. The 1348 bp cDNA contained an ORF of 1044 bp, which coded for a polypeptide of 347 amino acids. The deduced polypeptide had a predicted molecular weight of 38.3 kD and a pI of 5.77. Homology analysis demonstrated that, McChi5 protein, which contained a conserved domain of family 18 glycosyl hydrolyse, had the sequence similar to tobacco class V chitinases, several putative chitinases and chitinase-like proteins of Arabidopsis thiliania, and some chitinases from mammals, insects and bacteria. Southern blotting suggested that two copies of McChi5 gene and several homologous genes existed in the M. charatica genome. By RNA dot blotting analysis, expression of the McChi5 gene was detected in cotyledons, roots, stems, and leaves, and it was not induced by wounding treatment. The biological functions and the potential applications of Mochi5 gene were discussed.

Cloning and characterization of a balsam pear class I chitinase gene (Mcchit1) and its ectopic expression enhances fungal resistance in transgenic plants.

A balsam pear (Momordica charantia L.) chitinase (Mcchit1) was purified and sequenced at the N-terminal. The genomic and cDNA coding sequences of Mcchit1 were cloned by rapid amplification of 3' cDNA ends (3'-RACE) and the Y-shaped adaptor dependent extension (YADE) method. Sequence analysis showed that the Mcchit1 protein is a class I chitinase containing a chitin-binding domain and a catalytic domain, but no C-terminal extension. Northern blot indicated that the Mcchit1 transcription is wound-inducible. Overexpression of Mcchit1 dramatically increased intercellular and intracellular endochitinase activities, suggesting that the Mcchit1 gene encodes a secretory endochitinase. It was also found that overexpression of Mcchit1 significantly enhanced resistance to the plant pathogenic fungus Phytophthora nicotianae in transgenic N. benthamiana plants and against Verticillium wilt in transgenic cottons, indicating that the Mcchit1 gene can be a useful gene in plant engineering against fungal diseases.