ABSTRACT How protein signaling networks respond to different input strengths is an important but poorly understood problem in cell biology. For example, the small GTPase RhoA regulates both focal adhesion (FA) growth or disassembly, but whether RhoA serves as a switch selecting between cellular outcomes, or if outcomes are simply modulated by additional factors in the cell, is not clear. Here, we develop a photoswitchable RhoA guanine exchange factor, psRhoGEF, to precisely control endogenous RhoA activity. We also develop a FRET-based biosensor to allow visualization of RhoA activity together with psRhoGEF control. Using these new optical tools, we discover that low levels of RhoA activation preferentially induce FA disassembly in a Src-dependent manner, while high levels induce both FA growth and disassembly in a ROCK-dependent manner. Thus, rheostatic control of RhoA activation with photoswitchable RhoGEF reveals that cells can use signal amplitude to produce multiple responses to a single biochemical signal.
Rho GTPases play crucial roles in cell motility by regulation of actin cytoskeleton dynamics. For example, Cdc42 induces the formation of filopodia, and RhoA is involved in the assembly of actin stress fibers and cell contraction. While individual roles of RhoA and Cdc42 are well studied, cellular responses to combinations of RhoA and Cdc42 activity are poorly understood. Previous methods for investigating functions of multiple proteins, such as dominant-negative mutants or siRNA, may cause secondary effects, leading to non-specific pertubations throughout the cell. Here, we co-expressed single-chain photoswitchable activators of RhoA and Cdc42, psRhoGEF and psCdc42GEF, to control ratios of RhoA and Cdc42 activity with high spatiotemporal resolutions. Utilizing this method, we discover that simultaneous activation of RhoA and Cdc42 induces strong actin fibers at the edge of the cell, with a narrow range of ratios of RhoA/Cdc42 activation promoting the response. These cortical actin fibers are mediated by a Cdc42 effector MRCK, a RhoA effector ROCK, and mDia1, a convergent target of RhoA and Cdc42. Therefore, single-chain optobiochemical systems can dissect precise functions of combinations of multiple signaling molecules, which will be critical to understanding complex and dynamic cellular processes.
How protein signaling networks respond to different input strengths is an important but poorly understood problem in cell biology. For example, RhoA can promote focal adhesion (FA) growth or disassembly, but how RhoA activity mediates these opposite outcomes is not clear. Here, we develop a photoswitchable RhoA guanine nucleotide exchange factor (GEF), psRhoGEF, to precisely control endogenous RhoA activity. Using this optical tool, we discover that peak FA disassembly selectively occurs upon activation of RhoA to submaximal levels. We also find that Src activation at FAs selectively occurs upon submaximal RhoA activation, identifying Src as an amplitude-dependent RhoA effector. Finally, a pharmacological Src inhibitor reverses the direction of the FA response to RhoA activation from disassembly to growth, demonstrating that Src functions to suppress FA growth upon RhoA activation. Thus, rheostatic control of RhoA activation by psRhoGEF reveals that cells can use signal amplitude to produce multiple responses to a single biochemical signal.
Anthracnose, a destructive fungal disease, poses a significant threat to chili pepper (Capsicum annuum L.) production worldwide (de Silva et al. 2019). In South Korea, anthracnose outbreaks have traditionally been attributed to several Colletotrichum species such as C. gloeosporioides and C. acutatum. About 10% of the yield (chili production) is lost annually in South Korea due to chili anthracnose (Oo et al. 2020). During field surveys conducted in August 2017, symptomatic lesions resembling anthracnose were observed on chili pepper in two farmer's fields (Gochang and Cheongyang) in South Korea. Affected fruits exhibited characteristic symptoms, including circular sunken lesions with dark margins and abundant orange spore masses on the surface. About 20% of chili pepper fruit were affected in each field with an area of about 0.2 ha. Five putative Colletotrichum spp. isolates were obtained from six affected fruits (three from each field) following the procedure described by Cai et el. (2009). Three isolates (C01049, C01111, and C01115), representing each location, were selected to identify at the species level. Colonies on potato dextrose agar (incubated at 25°C in the dark for 7 days) were cottony with entire margins, white aerial mycelium and dark gray in the center. Conidia were hyaline, aseptate, cylindrical with bothnds round, and 17.8 - 30.5 × 6.0 -10.0 µm (mean 23.8 ×7.9 μm, n = 30). Appressoria were dark brown, irregular but mostly ovoid with smooth walls. These morphological features align with those of Colletotrichum spp. within the Colletotrichum gigasporum species (Liu et al. 2014). The identity of the pathogen was further confirmed through multi-locus phylogenetic analysis. The target genes including ITS, ACT, CHS-1, GAPDH, TUB2, and GS were amplified and sequenced using the primer sets ITS1/ITS4, ACT 512F/ ACT-783R, CHS-79F/ CHS-345R, GDF/GDR, T1/Bt2b, and GSF1/GSR1, respectively (Weir et al. 2012; Liu et al. 2014). The resulting sequences were deposited in GenBank (accession no: ITS: MT605261, MT605262, LC823714; ACT: MT612991, MT612992, LC823718; CHS-1: MT612993, MT612994, LC823717; GAPDH: LC811375, LC811376, LC823716; TUB2: MT612997, MT612998, LC823715; GS: LC811377, LC811378, LC823719). The constructed Bayesian and maximum likelihood tree based on combined sequences of ITS, ACT, CHS-1, GAPDH, TUB2, and GS confirmed the identification of the isolates (C01049, C01111, C01115) as C. gigasporum. Pathogenicity tests were conducted by inoculating healthy chili fruit with 70 µL of a conidial suspension (1×106 conidia /mL) of pure cultures of the isolates. The conidial suspension was applied on 10 wounded or 10 non-wounded fruit. The same number of fruit were treated with sterile distilled water as controls. Within 5 days of inoculation, symptoms consistent with anthracnose developed on the inoculated wounded fruit, whereas non-wounded and control fruit remained asymptomatic. This experiment was repeated twice. Colletotrichum gigasporum was re-isolated from diseased tissue of inoculated fruit. Colletotrichum gigasporum has been identified as the cause of anthracnose on Dalbergia odorifera, Carica papaya in China, and Brassica oleracea in India (Wan et al., 2018; Saini et al. 2022; He et al. 2023). To the best of our knowledge, this report marks the first documented instance of C. gigasporum causing anthracnose of chili pepper in South Korea. These results indicate that various species of Colletotrichum can be the fungi causing chili pepper anthracnose. The findings of this study emphasize the need for effective disease management strategies to mitigate impact of C. gigasporum on chili pepper cultivation in the region.
Chili pepper (Capsicum spp.) is an important crop in South Korea and is widely used in Korean cuisine, cultivated across a land area of roughly 29.8 thousand hectares, with a total production of 69 thousand tons (Lee et al., 2005; Statista, 2022). In September 2020, two farmer fields in Samcheok (37.444039°N, 129.135875°E; 37.633738°N, 128.911731°E), Gangwon province, South Korea, it is observed that chili pepper leaves showing yellowing and wilting symptoms, with an estimated disease incidence of approximately 10-15%. To identify the causal agents six affected plants were brought to lab. All the plants exhibited vascular discoloration in stem and root. After surface sterilizing small pieces of discolored tissue in 1% NaOCl for 30 s and rinsing twice in sterile distilled water, the tissue pieces were placed on water agar and incubated at 25°C for 10 days. Six pure isolates with consistent morphological characteristics were obtained by single spore isolation. Two representative isolates, NC17601 and NC20845 were selected for identification based morphological and molecular characters. Colonies on potato dextrose agar (PDA) during 10 days of incubation at 25°C in the dark were cottony white initially but progressively became dark as the formation of melanized microsclerotia. Conidiophores were hyaline, vertically branched or not, and had 2 - 4 phialides per node. Phialides were subulate and tapering from base to tip. The colonies produced abundant conidia, which were hyaline, single celled, smooth walled, cylindrical to oval, clustered on phialides, and 3.8 - 7.2 ×2.1 - 3.9 ㎛(mean ± SD: 5.2 ± 0.7 × 2.8 ± 0.5). Microsclerotia were aggregated form, various size and shape, and brown. These are the typical morphology of Verticillium dahliae (Inderbitiz et al. 2011; Yu et al. 2016). The molecular identification was later confirmed through PCR amplification, and sequencing targeting the translation elongation factor 1 alpha (TEF), actin (ACT), tryptophan synthase (TS), and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes using the primer sets described by Inderbitiz et al. (2011). The resulting sequences were deposited in GenBank with accession numbers LC761935 to LC761942. The maximum likelihood tree based on combined sequences of ACT, GPD, TEF and TS was inferred using RAxML- HPC2 on XSEDE as implemented on the CIPRES web server. The phylogenetic tree showed that the isolates were sit together with V. dahliae isolates (Ex-type PD322, PD227 and PD502). Pathogenicity tests using two isolates (NC17601 and NC20845) were conducted in the greenhouse, where 10 two weeks old seedlings per isolates (cv. Bigstar) were root-tip cut and then soaked in a fungal spore suspension of 107 conidia ml-1 for 1 h, while 10 seedlings were treated with sterile distilled water as a control. All the treated plants were maintained at 25°C (night)/ 25°C (Day) under natural light. After three weeks, all inoculated plants exhibit growth stunting with vascular discoloration in the stem and roots as compared to asymptomatic control plants. The isolates of V. dahliae were consistently re-isolated from discolored root tissues and identified based on morphological characteristics, thus fulfilling Koch's postulates. In South Korea, V. dahliae has been reported to cause wilt disease in various crops, including Kimchi cabbage and radish (Dumin et al. 2020; Choi et al. 2023). To the best of our knowledge, this is the first report that V. dahliae causing Verticillium wilt of chili pepper in South Korea. Overall, Verticillium dahliae is considered to be a significant threat to agriculture in South Korea, and efforts are being made to develop effective control strategies to mitigate its impact on crops.
Rho GTPases play crucial roles in cell motility by regulation of actin cytoskeleton dynamics. For example, Cdc42 induces the formation of filopodia, and RhoA is involved in the assembly of actin stress fibers and cell contraction. While individual roles of RhoA and Cdc42 are well studied, cellular responses to combinations of RhoA and Cdc42 activity are poorly understood. Previous methods for investigating functions of multiple proteins, such as dominant-negative mutants or siRNA, may cause secondary effects, leading to non-specific pertubations throughout the cell. Here, we co-expressed single-chain photoswitchable activators of RhoA and Cdc42, psRhoGEF and psCdc42GEF, to control ratios of RhoA and Cdc42 activity with high spatiotemporal resolutions. Utilizing this method, we discover that simultaneous activation of RhoA and Cdc42 induces strong actin fibers at the edge of the cell, with a narrow range of ratios of RhoA/Cdc42 activation promoting the response. These cortical actin fibers are mediated by a Cdc42 effector MRCK, a RhoA effector ROCK, and mDia1, a convergent target of RhoA and Cdc42. Therefore, single-chain optobiochemical systems can dissect precise functions of combinations of multiple signaling molecules, which will be critical to understanding complex and dynamic cellular processes.
Fig (Ficus carica L.) belonging to the Moraceae family is cultivated worldwide, with its primary production areas located in the Mediterranean region (Tous and Fergusen 1996). Yeongam-gun is a significant region for fig cultivation in Korea, accounting for 42% of the country's total fig cultivation area with approximately 1,400 fields (453ha, production yield 6000 tons). In July to November 2023, we observed severe rust disease in four fig orchards in Yeongam-gun (34°42'52.2"N, 126°31'32.16"E). The disease had affected 70% of the fig cultivation area (cv. Masui Dauphine). The area of each field is approximately 0.33~0.66ha, and 5 samples were collected from each field. Pustules of the pathogen were found in all samples. The diseased plants were deposited in the herbarium of the National Institute of Agricultural Sciences, Wanju, Korea (Specimen No. Cero_001). The initial symptoms were observed as chlorotic spots on the adaxial surface of the leaves, which developed into necrotic areas surrounded by chlorotic halos. Over time, dark brown spots were observed on the adaxial surface of the leaves, and abundant reddish-brown pustules were visible on the abaxial surface. Almost all leaves with these symptoms fell prematurely. Microscopic observation revealed that the urediniospores (n>50) were ellipsoidal, globose, obovoidal, or angular in shape, yellowish to faintly orange in color, sized 19.5–39.9 × 10.1–27.5 μm and had a wall thickness of 0.6–1.5 μm (average 1 μm). Telia were not observed. These morphological characteristics were comparable with that of Cerotelium fici (Gardner, 1997; Latinovic et al., 2015). For molecular analysis, genomic DNA was extracted from 3 to 5 samples, and the internal transcribed spacer region 2 (ITS2) and the large subunit (LSU) were amplified and sequenced using primer set: Rust2inv (Aime, 2006) and LR6 (Vilgalys and Hester, 1990). Furthermore, approximately 660 bp of the cytochrome c oxidase subunit III (COX3) gene was amplified and sequenced with CO3_F1 and CO3_R1 primers (Vialle et al., 2009). The obtained sequences were deposited in NCBI GenBank (GenBank accession no. PP491072 to PP491074 and PP491079 to PP491081). Phylogenetic tree analysis using the maximum likelihood method identified the isolate as Cerotelium fici. Pathogenicity test was conducted either by placing or by rubbing symptomatic leaf pieces on healthy leaves (5 leaves/plant) of three-year-olds fig plants (cv. Masui Dauphine) in a greenhouse in Wanju-gun, Jeonbuk special self-governing province. Healthy and symptom free leaf pieces were used for control. Two plants per treatment were used and the experiment repeated twice. The typical symptoms of fig rust disease were observed two weeks after inoculation in both methods. Control leaves were symptomless. C. fici was successfully reidentified from symptomatic tissues of inoculated leaves, fulfilling Koch’s postulates and confirmed as a causal agent of fig rust. Fig rust caused by C. fici has been reported in New Zealand, Montenegro, Hawaii and other tropical regions (McKenzie, 1986). To our knowledge, this is the first report of fig rust in Korea caused by Cerotelium fici. It is believed that this will be helpful in research on the management of fig rust disease.
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