Antimicrobial proteins (peptides) are known to play important roles in the innate host defense mechanisms of most living organisms, including plants, insects, amphibians and mammals. They are also known to possess potent antibiotic activity against bacteria, fungi, and even certain viruses. Recently, the rapid emergence of microbial pathogens that are resistant to currently available antibiotics has triggered considerable interest in the isolation and investigation of the mode of action of antimicrobial proteins (peptides). Plants produce a variety of proteins (peptides) that are involved in the defense against pathogens and invading organisms, including ribosome-inactivating proteins, lectins, protease inhibitors and antifungal peptides (proteins). Specially, the protease inhibitors can inhibit aspartic, serine and cysteine proteinases. Increased levels of trypsin and chymotrypsin inhibitors correlated with the plants resistance to the pathogen. Usually, the purification of antimicrobial proteins (peptides) with protease inhibitor activity was accomplished by salt-extraction, ultrafiltration and C18 reverse phase chromatography, successfully. We discuss the relation between antimicrobial and anti-protease activity in this review. Protease inhibitors from plants potently inhibited the growth of a variety of pathogenic bacterial and fungal strains and are therefore excellent candidates for use as the lead compounds for the development of novel antimicrobial agents.

Plant bZIP transcription factors play crucial roles in biological processes. In this study, 136 putative bZIP transcription members were identified in Brassica rapa. The bZIP family can be divided into nine groups according to the specific amino acid rich domain in B. rapa and Arabidopsis thaliana. To screen the cold stress responsive BrbZIP genes, we evaluated whether the transcription patterns of the BrbZIP genes were enhanced by cold treatment in the inbred lines, Chiifu and Kenshin, by microarray data analysis and qRT-PCR. The expression level of six genes increased significantly in Kenshin, but these genes were unchanged in Chiifu. These findings suggest that the six genes that encoded proteins containing N-rich regions might be involved in cold stress response. The results presented herein provide valuable information regarding the molecular basis of the bZIP transcription factors and their potential function in regulation growth and development, particularly in cold stress response.

Tomato is one of the major vegetable crops consumed worldwide. Tomato yellow leaf curl virus (TYLCV) and fungal Oidium sp. are devastating pathogens causing yellow leaf curl disease and powdery mildew. Such viral and fungal pathogens reduce tomato crop yields and cause substantial economic losses every year. Several commercial tomato varieties include Ty-5 (SlPelo) and Mildew resistance locus o 1 (SlMlo1) locus that carries the susceptibility (S-gene) factors for TYLCV and powdery mildew, respectively. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is a valuable genome editing tool to develop disease-resistant crop varieties. In this regard, targeting susceptibility factors encoded by the host plant genome instead of the viral genome is a promising approach to achieve pathogen resistance without the need for stable inheritance of CRISPR components. In this study, the CRISPR/Cas9 system was employed to target the SlPelo and SlMlo1 for trait introgression in elite tomato cultivar BN-86 to confer host-mediated immunity against pathogens. SlPelo-knockout lines were successfully generated, carrying the biallelic indel mutations. The pathogen resistance assays in SlPelo mutant lines confirmed the suppressed accumulation of TYLCV and restricted the spread to non-inoculated plant parts. Generated knockout lines for the SlMlo1 showed complete resistance to powdery mildew fungus. Overall, our results demonstrate the efficiency of the CRISPR/Cas9 system to introduce targeted mutagenesis for the rapid development of pathogen-resistant varieties in tomato.

A novel antifungal protein, M(r) = ca. 40 kDa, was isolated from pumpkin rind and designated Pr-1. When purified by anion exchange chromatography and HPLC, it inhibited growth of several fungi including Botrytis cinerea, Fusarium oxysporum, Fusarium solani and Rhizoctonia solani, as well as the yeast, Candida albicans, at 10-20 microM. It did not inhibit growth of Escherichia coli or Staphylococcus aureus even at 200 microM. Laser scanning microscopy of fungal cells exposed to rhodamine-labeled Pr-1 revealed that the protein accumulated and was localized on the cell surface. Uptake of the vital stain, SYTOX Green, was enhanced when fungal conidia were treated with Pr-1 suggesting that the protein has membrane permeabilization activity. Pr-1 was thermostable at 70 degrees C and did not lyse human red blood cells at 128 microM suggesting that the protein may be useful as an antifungal agent with little, if any human cytotoxicity.

A novel antifungal protein (Pr-2) was identified from pumpkin rinds using water-soluble extraction, ultrafiltration, cation exchange chromatography, and reverse-phase high-performance liquid chromatography. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry indicated that the protein had a molecular mass of 14865.57 Da. Automated Edman degradation showed that the N-terminal sequence of Pr-2 was QGIGVGDNDGKRGKR-. The Pr-2 protein strongly inhibited in vitro growth of Botrytis cinerea, Colletotrichum coccodes, Fusarium solani, Fusarium oxysporum, and Trichoderma harzianum at 10-20 microM. The results of confocal laser scanning microscopy and SYTOX Green uptake demonstrated that its effective region was the membrane of the fungal cell surface. In addition, this protein was found to be noncytotoxic and heat-stable. Taken together, the results of this study indicate that Pr-2 is a good candidate for use as a natural antifungal agent.