Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus
➤ Gửi thông báo lỗi ⚠️ Báo cáo tài liệu vi phạmNội dung chi tiết: Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus
Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus
bioRxiv preprint doi https .•■dci pra-10 1101-2021 11 26470170; this version posted November 27. 2021 Tl{which was not certihea by peer review) ts the Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus e author.lunder. All rights reserved. No reuse allcw< nups://Knoinuvien.comTwo Liberibacter Effectors Combine to Suppress Critical Innate Immune Defenses andFacilitate Huanglongbing Pathogenesis in CitrusSupratim Basil5. Loan Huynh5. Shujian Zhang5, Roel Rabara5, Hau Nguyen5. Jeanette Valesquez5.Gui Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus xia Hao’. Godfrey Miles’. Qingchun Shi85. Ed Stover, and Goutam Gupta5’5Biolab. New Mexico Consortium, 100 Entrada Drive. Los Alamos, NM 87544. USA=u.Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus
s. Horticultural Research Laboratory 2001 South Rock Road Fort Pierce. FL 34945. USA♦Correspondence: ggupta@newmexicoconsonium.orgbioRxỉv preprint dobioRxiv preprint doi https .•■dci pra-10 1101-2021 11 26470170; this version posted November 27. 2021 Tl{which was not certihea by peer review) ts the Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus rights reserved. No reuse allow nnps://Knotnuvien.comAbstractGenome sequence analyses predicted the presence of effectors in the gram-negative Candidatus Liberibacter asiaticus (CLas) even without the presence of a classical type III secretion system. Since CLas is not culturable, it is not possible Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus to perform traditional gene knockout experiments to determine the role of various effectors in Huanglongbing (HLB) pathogenesis. Therefore, we followTwo liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus
ed an alternative functional genomics approach to examine the role of the CLas effectors in HLB pathogenesis in general and more specifically in supprbioRxiv preprint doi https .•■dci pra-10 1101-2021 11 26470170; this version posted November 27. 2021 Tl{which was not certihea by peer review) ts the Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus studies by LC-MS/MS were conducted to screen the putative interacting citrus protein partners of P235 and Effector 3 from the healthy and (Las-infected Hamlin extracts and the most probable candidates were identified based upon their high protein scores from LC-MS/MS. Second, a transgenic tobacco sp Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus lit GFP system was designed for in planta detection of the most probable citrus interacting protein partners of P235 and Effector 3. Third, in vitro aTwo liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus
nd in planta studies were performed to show that each of two effectors interacts with and inhibits the functions of multiple citrus proteins belongingbioRxiv preprint doi https .•■dci pra-10 1101-2021 11 26470170; this version posted November 27. 2021 Tl{which was not certihea by peer review) ts the Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus ding protein (LTP). and induction of premature programmed cell death (PCD), thereby supporting CLas infection and HLB pathogenesis. Finally, an LTP mimic was designed to sequester and block the (Las effector and to rescue the bactericidal activity of LTP.Key words: effectors, immunity, modelling, RO Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus S, PAMPIntroductionHuanglongbing (HLB) is the most devastating citrus disease While endemic in Asia for over a century3-4, HLB was first encountered aTwo liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus
bout a 15 years ago in Florida with the emergence of Asian Citrus Psyllid (ACP) vectors carrying HLB-causing Candidates Liberibacter asiaticus (CLas).bioRxiv preprint doi https .•■dci pra-10 1101-2021 11 26470170; this version posted November 27. 2021 Tl{which was not certihea by peer review) ts the Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus HLB management tools are urgently needed for sustaining a productive and profitable citnis industry5. These tools include development of both bactericidal and anti-infective molecules for HLB treatment. Recently, we reported the development of novel citrus-derived CLas-kiUer peptides that can be use Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus d for HLB treatment bybioRxỉv preprint doi https/rơci ọra'10.1101'2021 11 26 470170; this version posted November 27.2021 Tl(which was not certified bTwo liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus
y peer review) IS the autncr/tunder. All rights reserved. No reuse altowi nups://Knoinuvien.comtopical delivery'6. Ill this study, we focused first onbioRxiv preprint doi https .•■dci pra-10 1101-2021 11 26470170; this version posted November 27. 2021 Tl{which was not certihea by peer review) ts the Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus therapeutic and anti-infective molecules to block them. Typically, the plant innate immune defense involves multiple pathways including pathogen or microbe-associated molecular pattern (PAMP or MAMP) triggered immunity (PTI or MTI), effector triggered immunity (ETI). and plant hormone, such as sali Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus cylic (SA), jasmonic acid (JA), and ethylene (ET) induced immunity7*12. The PTI or MTI provides the first line of plant defense against pathogens or mTwo liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus
icrobes through the recognition of PAMP or MAMP. such as bacterial liposaccharide (LPS). elongation factor thermal unstable (EF-Tu). flagellin. PAMP obioRxiv preprint doi https .•■dci pra-10 1101-2021 11 26470170; this version posted November 27. 2021 Tl{which was not certihea by peer review) ts the Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus (FLS2). EF-Tu receptor (EFR). The plasma membrane PRR recognition induces intracellular mitogen-associated protein kinase (MAPK) signaling leading to the expression of pathogen-related (PR) or defense genes13'15. However, pathogen effectors can block both intracellular and extracellular steps in th Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus e PTI pathway 16,1?. To counter pathogen induced blocking of the PTI pathway, plants have evolved the ETI pathway in which intracellular nod-like receTwo liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus
ptors (NLR) recognize the pathogen effectors and augment the MAPK signaling and PR gene expression. The ETI pathway also induces hypersensitive responbioRxiv preprint doi https .•■dci pra-10 1101-2021 11 26470170; this version posted November 27. 2021 Tl{which was not certihea by peer review) ts the Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus ETI pathways also couple to intracellular plant hormone SA/JA/ET pathways, which also involve ROS production and induction of PR genes. It has been demonstrated that the effectors from plant pathogenic bacteria can inhibit one or more steps in these pathways 1S*21. Also, the bacterial effectors are Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus known to subvert multiple steps leading to programmed cell death (PCD) in plant, which is a form of immune defense by PTI and/or ETI to control infecTwo liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus
tion 22*24. Therefore, it was of interest for US to determine which steps in the citrus innate immune defense are affected by the CLas effectors. NotebioRxiv preprint doi https .•■dci pra-10 1101-2021 11 26470170; this version posted November 27. 2021 Tl{which was not certihea by peer review) ts the Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus type III or VI secretion system like many other plant pathogenic gram-negative bacteria •7’28. Gram-negative bacteria with 5 Mb genomes have several hundred unique effectors 29 as opposed to only 20 effectors identified, so far. from CLas3013However, interactome studies revealed that even an effecto Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus r from a gram-negative bacterium carrying hundreds of effectors can target more than one protein from the host plant r-32. Therefore, it was of intereTwo liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus
st to examinebioRxN preprint doi https .''del <ỵg.'10.1101’2021 11 26 470170; this version posted November 27, 2021 Tlrn,u^.4.u.(which was not certifibioRxiv preprint doi https .•■dci pra-10 1101-2021 11 26470170; this version posted November 27. 2021 Tl{which was not certihea by peer review) ts the Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus s proteins to effectively suppress the innate immune defense and establish infection.In this study, we focused on two CLas effectors. P235 and Effector 3. First, we performed in vitro and in planta studies to identify the prominent citrus proteins targeted by P235 and Effector 3. Second, we performe Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus d functional assays to determine whether P235 and Effector 3 have inhibitory effects on their citrus protein targets. Next, we performed molecular dynTwo liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus
amic simulations to analyze the details of interaction between P235 (and Effector 3) and their selected citrus targets and predicted which pairwise inbioRxiv preprint doi https .•■dci pra-10 1101-2021 11 26470170; this version posted November 27. 2021 Tl{which was not certihea by peer review) ts the Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus ic mutations on the citrus protein(s) that affect the critical pairwise interactions. We discovered that each of the two effectors can directly target several citrus proteins, which belong to the innate immune networks. A clear understanding of the inhibitory mechanisms will provide guidelines for c Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus ountering CLas effectors and developing anti-infectives to block HLB pathogenesis.MethodsExperimental ProceduresPlant Materials and growth conditions.Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus
Hamlin trees verified as being HLB-free and ACP-friendly were purchased and placed in the green house. One branch cage placed in the upper part of eabioRxiv preprint doi https .•■dci pra-10 1101-2021 11 26470170; this version posted November 27. 2021 Tl{which was not certihea by peer review) ts the Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus insects were allowed to feed on the trees for a week and then the insects were killed by spraying with topical insecticide. The ACPs were tested for C.Las and the trees were subsequently returned to the greenhouse. Leaf samples were collected from the untreated and infected plants and flash frozen i Two liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus n liquid nitrogen and stored for further analysis.Cloning and overexpression of effectors and targets in E. coli. The effectors from Liberibacter asiaTwo liberibacter effectors combine to suppress critical innate immune defenses and facilitate huanglongbing pathogenesis in citrus
ticus were identified, codon optimized and cloned in pUC57 by GenScript. The effectors were then amplified and cloned in pET28(a) vector between NdelGọi ngay
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