The 35S:CsLOB1‐GR construct contains green fluoresent protein (GFP) and neomycin phosphotransferase II (NptII) to facilitate the screening of transgenic plants. (A) Diagram of the 35S:CsLOB1‐GR plasmid used to construct CsLOB1 transgenic plants. Scale bar represents 10 μm.Įctopic CsLOB1 expression in citrus induces pustule formation. The blue colour indicates the nuclear localization stained by Hoechst. The green colour indicates the fluorescence of enhanced yellow fluorescent protein (EYFP).
The fluorescence figures were taken 10 min after Hoechst dye addition to the protoplast at the rate of 1 : 600. CsLOB1 localization to the nucleus was confirmed by co‐localization with Hoechst 33342. 35S:EYFP‐CsLOB1 was transformed into citrus protoplast via polyethylene glycol (PEG)‐calcium‐mediated transfection. All tissues in each replicate are from the same plant. YL, young leaves, 15 days after flushing ML, mature leaves, over 30 days after flushing FL, folded flowers ST, stems. Each value represents the mean ± standard deviation (SD) of three replicates. The endogenous housekeeping gene used was GAPDH (Cs2g14940, glyceraldehyde 3‐phosphate dehydrogenase). (A) Expression pattern of the CsLOB1 gene in different tissues of Duncan grapefruit. Tissue expression pattern and subcellular localization analyses of the CsLOB1 gene. This study advances our understanding of the function of CsLOB1 and the molecular mechanism of how Xcc causes canker symptoms via CsLOB1.Ĭ3HC4-type RING finger protein CsLOB1 Xanthomonas citrus canker susceptibility gene. Cs2g20600, which encodes a zinc finger C3HC4-type RING finger protein, has been identified to be a direct target of CsLOB1. As CsLOB1 acts as a transcriptional factor, we identified putative targets of CsLOB1 via bioinformatic and electrophoretic mobility shift assays. Twelve citrus genes that are induced by PthA4 are also stimulated by the DEX-induced expression of CsLOB1. When CsLOB1-GR-transgenic Duncan grapefruit leaves were inoculated with Xcc306ΔpthA4 and treated with DEX, typical canker symptoms, including hypertrophy, hyperplasia and water soaking symptoms, were observed on DEX-treated transgenic plant leaves, but not on mock-treated plants. Weaker water soaking was observed with pustule formation in CsLOB1-GR transgenic plants following DEX treatment. Water soaking is typically associated with symptoms of citrus canker. Pustule formation was not observed in DEX-treated wild-type plants and in non-treated CsLOB1-GR transgenic plants. We showed that CsLOB1 expression driven by dexamethasone (DEX) in CsLOB1-GR transgenic plants is associated with pustule formation following treatment with DEX. Subcellular localization analysis of CsLOB1 protein in citrus protoplast revealed that CsLOB1 is primarily localized in the nucleus. Here, we report our recent progress in the functional characterization of CsLOB1. PthA, a transcriptional activator-like (TAL) effector, directs the expression of the canker susceptibility gene CsLOB1. citri (Xcc) is an important plant-pathogenic bacterium that causes citrus canker disease worldwide.
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They have a nice building and a good location, but there's no direction on how to get tasks done and no way of ensuring that you're able to get them done efficiently.Xanthomonas citri ssp.
Micromanaging is huge here no one trusts you to do your job so every 2 minutes you'll be asked what you're working on and what you're doing just in case you're doing something like not working in some way. As a new person you'll find yourself swamped with a ton of work, most of which was left by the people who had the job before you and just never bothered to finish it or perhaps they didn't have time, because every time I started a task the person who was supposedly training me would stop me and hand me something else to do, so I never actually finished anything I was given.
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