CaWRKY50 Acts as a Negative Regulator in Response to Colletotrichum scovillei Infection in Pepper.

Chili anthracnose is one of the most common and destructive fungal pathogens that affects the yield and quality of pepper. Although WRKY proteins play crucial roles in pepper resistance to a variety of pathogens, the mechanism of their resistance to anthracnose is still unknown. In this study, we found that CaWRKY50 expression was obviously induced by Colletotrichum scovillei infection and salicylic acid (SA) treatments. CaWRKY50-silencing enhanced pepper resistance to C. scovillei, while transient overexpression of CaWRKY50 in pepper increased susceptibility to C. scovillei. We further found that overexpression of CaWRKY50 in tomatoes significantly decreased resistance to C. scovillei by SA and reactive oxygen species (ROS) signaling pathways. Moreover, CaWRKY50 suppressed the expression of two SA-related genes, CaEDS1 (enhanced disease susceptibility 1) and CaSAMT1 (salicylate carboxymethyltransferase 1), by directly binding to the W-box motif in their promoters. Additionally, we demonstrated that CaWRKY50 interacts with CaWRKY42 and CaMIEL1 in the nucleus. Thus, our findings revealed that CaWRKY50 plays a negative role in pepper resistance to C. scovillei through the SA-mediated signaling pathway and the antioxidant defense system. These results provide a theoretical foundation for molecular breeding of pepper varieties resistant to anthracnose.

Magnesium transporter CsMGT10 of tea plants plays a key role in chlorosis leaf vein greening.

Magnesium (Mg2+), as the central atom of chlorophyll, is the most abundant divalent cation for plant growth and development in living cells. MRS2/MGT magnesium transporters play important roles in coping with magnesium stress, chloroplast development and photosynthesis. However, the molecular mechanism of MGT influencing tea plant leaf vein color remains unknown. Here, we demonstrate that CsMGT10 may be a potential transporter influencing leaf vein color. CsMGT10 belongs to Clade A member of MRS2/MGT family. CsMGT10 has the highest expression level in leaves of tea plants. And it is mainly expressed in aboveground parts, especially in vascular bundles. Moreover, CsMGT10 localizes to the chloroplast envelope of tea plants with a high affinity to Mg2+. And the GMN motif is required for its magnesium transport function. Ectopic expression of CsMGT10 in Arabidopsis leaf variegation mutant var5-1 can restore green color of chlorosis leaf veins, and the contents of chlorophyll and carotenoid change significantly, proving its essential role in leaf vein greening. Furthermore, the chlorophyll and carotenoid of tea leaves treated with CsMGT10 antisense oligonucleotides also decrease significantly. Our findings indicate that CsMGT10 mainly acts as Mg2+ transporter in chloroplast envelope of leaf veins, which may play a key role in leaf vein greening of tea plants.

First Report of Colletotrichum gloeosporioides Causing Anthracnose on Pepper in Shaanxi Province, China.

Pepper (Capsicum annuum L.) is an important solanaceous vegetable crop, with high nutritional and economic value. However, it is susceptible to Colletotrichum spp. infection during its growth and development, which seriously affects production yield and quality. Chili anthracnose, caused by Colletotrichum spp., is one of the most destructive diseases of pepper. In August 2020, chili anthracnose was observed with widespread distribution in the horticulture field of Northwest A&F University (34.16° N, 108.04° E) in Shaanxi Province, China. Approximately 60% of the pepper plants had disease symptoms typical of anthracnose. Lesions on pepper fruits were dark, circular, sunken, and necrotic, with the presence of orange to pink conidial masses (Figure S1A). To perform fungal isolation, the tissue at the lesion margin was cut from eight symptomatic fruits, surface disinfested with 75% ethanol for 30 s, and 2% NaClO for 1 min, then rinsed three times with sterile distilled water and dried on sterile filter paper. The tissues were placed on potato dextrose agar (PDA) and incubated at 28 ºC in the dark. After 3 days, hyphae growing from tissue of each lesion were recultured on PDA (Liu et al. 2016). A representative single-spore isolate (NWAFU2) was used for morphological characterization, molecular analysis, phylogenetic analysis, and pathogenicity tests. NWAFU2 colonies had gray-white aerial mycelium, and the reverse side of the colonies was dark gray to light yellow after 10-days growth on PDA (Figure S1B-C). Conidia were cylindrical, aseptate, with obtuse to slightly rounded ends, and measured 10.1 to 16.9 (length) × 4.7 to 7.0 (width) µm (n=50) (Figure S1D). Based on morphological features, the isolate was consistent with the description of C. gloeosporioides species complex (Weir et al. 2012). For molecular identification, genomic DNA was extracted using a CTAB method and the internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and partial sequences of actin (ACT) genes were amplified and sequenced using primers ITS1F/ITS4, GDF1/GDR1 and ACT-512F/ACT-783R, respectively (Dowling et al. 2020). Using the BLAST, ITS, ACT, GAPDH gene sequences (GenBank accession nos. MW258690, MW258691 and MW258692, respectively) were 100%, 100% and 98.19% identical to ZJL-4 of C. gloeosporioides (GenBank accession nos. MN075757, MN058142 and MN075666, respectively). Phylogenetic analysis was conducted using MEGA-X (Version 10.0) based on the concatenated sequences of published ITS, ACT and GAPDH for Colletotrichum species using Neighbor-Joining algorithm. The identified isolate (NWAFU2) was closely related to C. gloeosporioides (Figure S2). To confirm the pathogenicity, ten healthy pepper fruits were surface-sterilized and 2 µL of conidial suspension (1×106 conidia/mL) was injected the surface of pepper. Five fruits were inoculated with 2µL sterile distilled water as controls. After inoculation, the fruits were kept in a moist chamber at 28°C in the dark. The experiment was repeated three times. Anthracnose symptoms similar to those observed in the field, were observed 7 days after inoculation (Figure S1F) and control fruits remained healthy. A similarly inoculated detached leaf assay resulted in water-soaked lesions 3 days after inoculation. C. gloeosporioides was reisolated from the infected pepper fruits, fulfilling Koch's postulates. C. gloeosporioides has been reported to cause chili anthracnose in Sichuan Province, China (de Silva et al. 2019; Liu et al. 2016). However, Shaanxi is one of the main pepper producing areas in china and it is geographically distinct from Sichuan; its climate and environmental conditions are different from Sichuan. Knowledge that C. gloeosporioides causes chili anthracnose of pepper in Shaanxi province, China may aid in the selection of appropriate management tactics for this disease. Reference: de Silva, D. D., Groenewald, J. Z., Crous, P. W., Ades, P. K., Nasruddin, A., Mongkolporn, O., and Taylor, P. W. J. 2019. Identification, prevalence and pathogenicity of Colletotrichum species causing anthracnose of Capsicum annuum in Asia. IMA Fungus 10:8. Dowling, M., Peres, N., Villani, S., and Schnabel, G. 2020. Managing Colletotrichum on Fruit Crops: A "Complex" Challenge. Plant Dis 104:2301-2316. Liu, F. L., Tang, G. T., Zheng, X. J., Li, Y., Sun, X. F., Qi, X. B., Zhou, Y., Xu, J., Chen, H. B., Chang, X. L., Zhang, S. R., and Gong, G. S. 2016. Molecular and phenotypic characterization of Colletotrichum species associated with anthracnose disease in peppers from Sichuan Province, China. Sci Rep 6. Weir, B. S., Johnston, P. R., and Damm, U. 2012. The Colletotrichum gloeosporioides species complex. Stud Mycol 73:115-180.