Background: Effector proteins function not only as toxins to induce plant cell death, but also enable pathogens to suppress or evade plant defense responses. NLP-like proteins are considered to be effector proteins, and they have been isolated from bacteria, fungi, and oomycete plant pathogens. There is increasing evidence that NLPs have the ability to induce cell death and ethylene accumulation in plants. Results: We evaluated the expression patterns of 11 targeted PcNLP genes by qRT-PCR at different time points after infection by P. capsici. Several PcNLP genes were strongly expressed at the early stages in the infection process, but the expression of other PcNLP genes gradually increased to a maximum at late stages of infection. The genes PcNLP2, PcNLP6 and PcNLP14 showed the highest expression levels during infection by P. capsici. The necrosis-inducing activity of all targeted PcNLP genes was evaluated using heterologous expression by PVX agroinfection of Capsicum annuum and Nicotiana benthamiana and by Western blot analysis. The members of the PcNLP family can induce chlorosis or necrosis during infection of pepper and tobacco leaves, but the chlorotic or necrotic response caused by PcNLP genes was stronger in pepper leaves than in tobacco leaves. Moreover, PcNLP2, PcNLP6, and PcNLP14 caused the largest chlorotic or necrotic areas in both host plants, indicating that these three genes contribute to strong virulence during infection by P. capsici. This was confirmed through functional evaluation of their silenced transformants. In addition, we further verified that four conserved residues are putatively active sites in PcNLP1 by site-directed mutagenesis. Conclusions: Each targeted PcNLP gene affects cells or tissues differently depending upon the stage of infection. Most PcNLP genes could trigger necrotic or chlorotic responses when expressed in the host C. annuum and the non-host N. benthamiana. Individual PcNLP genes have different phytotoxic effects, and PcNLP2, PcNLP6, and PcNLP14 may play important roles in symptom development and may be crucial for virulence, necrosis-inducing activity, or cell death during infection by P. capsici.

Colletotrichum is a cosmopolitan fungal genus, and its species are known for their important role as plant pathogens. They also occur as endophytes and saprobes. The genus comprises species complexes, many of which include cryptic species that are morphologically similar but phylogenetically distinct. Colletotrichum is characterized by globose to subglobose ascomata and acervular conidiomata and hyaline, aseptate ascospores and conidia, although a few species produce septate ascospores. Ascospores are typically cylindrical, oval, or fusiform, while conidia are mostly ellipsoidal to ovoid. Many Colletotrichum species also produce appressoria, which are formed at the tips of germ tubes or directly from the mycelium. In this study, we collected diseased leaves and pods with spots, blight, and blotches from Chiang Mai, Chiang Rai, and Tak provinces in Thailand. For species identification and delimitation, we employed a biphasic approach incorporating morphological data and multigene phylogenetic analyses of the locus of the internal transcribed spacer (ITS, nuclear rDNA consisting of ITS1-5.8S-ITS2) and the genes encoding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS1), actin (ACT), beta-tubulin 2 (TUB2), histone (H3), and calmodulin (CAM). We obtained 20 Colletotrichum isolates representing eight species, among which we propose two new species: Colletotrichum dendrobii (from Dendrobium sp.) and C. musichiangmaiense (from Musa sp.). We also establish 13 new host records (for six species): C. castaneae (on Jasminum grandiflorum), C. chrysophilum (on Terminalia sp.), C. endophyticum (on Artocarpus heterophyllus, Begonia luxurians, Cassia sp., Castanopsis sp., Ficus auriculata, and Schefflera sp.), C. fructicola (on Castanea sp., Hedychium sp., and Rhododendron sp.), C. jiangxiense (on Artocarpus sp.), and C. schimae (on Jasminum sp.). In addition, this is the first study to present the sexual morph of C. endophyticum.

Background: Pectate lyases (PELs) play an important role in the infection process of plant pathogens and also have a commercial significance in industrial applications. Most of the PELs were expressed as soluble recombinant proteins, while a few recombinant proteins were insoluble. The production of a large-scale soluble recombinant PEL would allow not only a more detailed structural and functional characterization of this enzyme but also may have important applications in the food industry. Results: We cloned a new pectate lyase gene (Pcpel2) from Phytophthora capsici. Pcpel2 was constructed by pET system and pMAL system, and both constructs were used to express the PCPEL2 in Escherichia coli BL21 (DE3) pLysS. The expressed products were purified using affinity chromatography and gel filtration chromatography. The purity, specific activity and pathogenicity of the purified PCPEL2 expressed by the pMAL system were higher than the purified PCPEL2 expressed by the pET system. In addition, some other characteristics of the purified PCPEL2 differed from the two systems, such as crystallographic features. Purified PCPEL2 expressed by the pMAL system was crystallized by the hanging-drop vapour-diffusion method at 289 K, and initial crystals were grown. Conclusion: The two different methods and comparison presented here would be highly valuable in obtaining an ideal enzyme for the downstream experiments, and supply an useful alternative to purify some insoluble recombinant proteins.