Functional Evaluation of the Signal Peptides of Secreted Proteins

Yin, Wei-Xiao; Wang, Yufu; Chen, Tao; Lin, Yang; Luo, Chaoxi

doi:10.21769/bioprotoc.2839

Cited by 60 publications

(51 citation statements)

References 6 publications

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“…This SP has been proven to have secretion function in yeast ( Dou et al, 2008 ). pSUC2: Mg-PEL1(SP)-Invertase and pSUC2:Avr1b (SP)-Invertase were transformed into the yeast strain YTK12 by the lithium acetate method ( Gietz and Schiestl, 2007 ; Yin et al, 2018 ). The yeast strain YTK12 and YTK12 carrying pSUC2 empty vector were used as negative controls.…”

Section: Methodsmentioning

confidence: 99%

A Meloidogyne graminicola Pectate Lyase Is Involved in Virulence and Activation of Host Defense Responses

Chen

Lin

et al. 2021

Front. Plant Sci.

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Plant-parasitic nematodes secrete an array of cell-wall-degrading enzymes to overcome the physical barrier formed by the plant cell wall. Here, we describe a novel pectate lyase gene Mg-PEL1 from M. graminicola. Quantitative real-time PCR assay showed that the highest transcriptional expression level of Mg-PEL1 occurred in pre-parasitic second-stage juveniles, and it was still detected during the early parasitic stage. Using in situ hybridization, we showed that Mg-PEL1 was expressed exclusively within the subventral esophageal gland cells of M. graminicola. The yeast signal sequence trap system revealed that it possessed an N-terminal signal peptide with secretion function. Recombinant Mg-PEL1 exhibited hydrolytic activity toward polygalacturonic acid. Rice plants expressing RNA interference vectors targeting Mg-PEL1 showed an increased resistance to M. graminicola. In addition, using an Agrobacterium-mediated transient expression system and plant immune response assays, we demonstrated that the cell wall localization of Mg-PEL1 was required for the activation of plant defense responses, including programmed plant cell death, reactive oxygen species (ROS) accumulation and expression of defense-related genes. Taken together, our results indicated that Mg-PEL1 could enhance the pathogenicity of M. graminicola and induce plant immune responses during nematode invasion into plants or migration in plants. This provides a new insight into the function of pectate lyases in plants-nematodes interaction.

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Section: Methodsmentioning

confidence: 99%

A Meloidogyne graminicola Pectate Lyase Is Involved in Virulence and Activation of Host Defense Responses

Chen

Lin

et al. 2021

Front. Plant Sci.

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“…To create the vectors used for signal peptide functional analysis, the signal peptide sequences of the secreted cysteine proteases were cloned from P. parasitica Pp016 genomic DNA and inserted into the Eco RI/ Xho I restriction sites of pSUC2 (Jacobs et al , ; Yin et al , ) using a ClonExpress II One Step Cloning Kit (Vazyme).…”

Section: Methodsmentioning

confidence: 99%

“…Functional evaluation of the predicted signal peptide sequences was performed according to a previously published protocol (Yin et al , ). Briefly, the sequence‐verified pSUC2 constructs were transformed into yeast YTK12 competent cells.…”

Section: Methodsmentioning

confidence: 99%

“…Step Cloning Kit (Vazyme). The vector was digested with NotI restriction enzyme (NEB), and the smaller fragment was inserted into the binary vector pART27 (Gleave, 1992) To create the vectors used for signal peptide functional analysis, the signal peptide sequences of the secreted cysteine proteases were cloned from P. parasitica Pp016 genomic DNA and inserted into the EcoRI/XhoI restriction sites of pSUC2 (Jacobs et al, 1997;Yin et al, 2018) using a ClonExpress II One Step Cloning Kit (Vazyme).…”

Section: Onementioning

confidence: 99%

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Two Phytophthora parasitica cysteine protease genes, PpCys44 and PpCys45, trigger cell death in various Nicotiana spp. and act as virulence factors

Zhang

Yang

et al. 2020

Molecular Plant Pathology

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Proteases secreted by pathogens have been shown to be important virulence factors modifying plant immunity, and cysteine proteases have been demonstrated to participate in different pathosystems. However, the virulence functions of the cysteine proteases secreted by Phytophthora parasitica are poorly understood. Using a publicly available genome database, we identified 80 cysteine proteases in P. parasitica, 21 of which were shown to be secreted. Most of the secreted cysteine proteases are conserved among different P. parasitica strains and are induced during infection. The secreted cysteine protease proteins PpCys44/45 (proteases with identical protein sequences) and PpCys69 triggered cell death on the leaves of different Nicotiana spp. A truncated mutant of PpCys44/45 lacking a signal peptide failed to trigger cell death, suggesting that PpCys44/45 functions in the apoplastic space. Analysis of three catalytic site mutants showed that the enzyme activity of PpCys44/45 is required for its ability to trigger cell death. A virus‐induced gene silencing assay showed that PpCys44/45 does not induce cell death on NPK1 (Nicotiana Protein Kinase 1)‐silenced Nicotiana benthamiana plants, indicating that the cell death phenotype triggered by PpCys44/45 is dependent on NPK1. PpCys44‐ and PpCys45‐deficient double mutants showed decreased virulence, suggesting that PpCys44 and PpCys45 positively promote pathogen virulence during infection. PpCys44 and PpCys45 are important virulence factors of P. parasitica and trigger NPK1‐dependent cell death in various Nicotiana spp.

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“…The transformed colonies were transferred to fresh CMD-W and YPRAA (1% yeast extract, 2% peptone, 2% raffinose and 2 mg/ mL antimycin A) plates and incubated at 30 ℃. The enzymatic activity of invertase was detected by the reduction of TTC to insoluble red-coloured triphenylformazan as described previously [41]. The yeast transformants were inoculated in 2 to 3 mL of YPDA (1% yeast extract, 2% peptone, 2% glucose, 0.03% adenine hemisulfate and 2% agar) medium and incubated for 24-36 h at 30 ℃.…”

Section: Validation Of Predicted Signal Peptidementioning

confidence: 99%

Identification and characterization of the Nep1-like protein UvNLP in rice false smut fungus Ustilaginoidea virens

Wang¹,

Qu²,

Liu³

et al. 2020

Preprint

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Background The necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are a broadly distributed superfamily of protiens in plant-associated microorganisms, including bacteria, fungi, and oomycetes. NLPs are considered to be important virulence factors, and some have been well studied. However, the role of NLPs in Ustilaginoidea virens , the agent of rice false smut fungus, remains to be uncovered.Results A protein containing NLP-specific NPP1 domain was identified in U. virens and named UvNLP. Phylogenetic analysis revealed that UvNLP was a type 2 NLP, and real-time PCR revealed that UvNLP was highly expressed at 3 days post-inoculation. A yeast secretion assay demonstrated that UvNLP contained a functional signal peptide (SP). Furthermore, transient expression of UvNLP in Nicotiana benthamiana induced weak cell death, and replacement of the SP enhanced cell death. Real-time PCR indicated that UvNLP induced the expression of plant defence related genes.Conclusions We identified a secreted protein UvNLP in U. virens that was infection related and could induce plant cell death and defence responses. This study provided evidence that NLPs act as proteinaceous MAMPs, giving insight into the elucidation of the pathogenicity mechanism of U. virens. The UvNLP might be used as a potential plant defence inducer for disease control in practice.

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Functional Evaluation of the Signal Peptides of Secreted Proteins

Cited by 60 publications

References 6 publications

A Meloidogyne graminicola Pectate Lyase Is Involved in Virulence and Activation of Host Defense Responses

A Meloidogyne graminicola Pectate Lyase Is Involved in Virulence and Activation of Host Defense Responses

Two Phytophthora parasitica cysteine protease genes, PpCys44 and PpCys45, trigger cell death in various Nicotiana spp. and act as virulence factors

Identification and characterization of the Nep1-like protein UvNLP in rice false smut fungus Ustilaginoidea virens

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Functional Evaluation of the Signal Peptides of Secreted Proteins

Cited by 60 publications

References 6 publications

A Meloidogyne graminicola Pectate Lyase Is Involved in Virulence and Activation of Host Defense Responses

A Meloidogyne graminicola Pectate Lyase Is Involved in Virulence and Activation of Host Defense Responses

Two Phytophthora parasitica cysteine protease genes, PpCys44 and PpCys45, trigger cell death in various Nicotiana spp. and act as virulence factors

Identification and characterization of the Nep1-like protein UvNLP in rice false smut fungus Ustilaginoidea virens﻿

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Identification and characterization of the Nep1-like protein UvNLP in rice false smut fungus Ustilaginoidea virens