scholarly journals Functional Characterization of the Nep1-Like Protein Effectors of the Necrotrophic Pathogen – Alternaria brassicae

2021 ◽  
Vol 12 ◽  
Author(s):  
Deepak Duhan ◽  
Shivani Gajbhiye ◽  
Rajdeep Jaswal ◽  
Ravindra Pal Singh ◽  
Tilak Raj Sharma ◽  
...  
Keyword(s):  
Cell Death ◽  
Host Species ◽  
Functional Characterization ◽  
Subcellular Location ◽  
Ethylene Biosynthesis ◽  
Alternaria Brassicae ◽  
Necrotrophic Pathogen ◽  
Molecular Pattern ◽  
Pathogen Associated Molecular Pattern

Alternaria brassicae is an important necrotrophic pathogen that infects the Brassicaceae family. A. brassicae, like other necrotrophs, also secretes various proteinaceous effectors and metabolites that cause cell death to establish itself in the host. However, there has been no systematic study of A. brassicae effectors and their roles in pathogenesis. The availability of the genome sequence of A. brassicae in public domain has enabled the search for effectors and their functional characterization. Nep1-like proteins (NLPs) are a superfamily of proteins that induce necrosis and ethylene biosynthesis. They have been reported from a variety of microbes including bacteria, fungi, and oomycetes. In this study, we identified two NLPs from A. brassicae viz. AbrNLP1 and AbrNLP2 and functionally characterized them. Although both AbrNLPs were found to be secretory in nature, they localized differentially inside the plant. AbrNLP2 was found to induce necrosis in both host and non-host species, while AbrNLP1 could not induce necrosis in both species. Additionally, AbrNLP2 was shown to induce pathogen-associated molecular pattern (PAMP)-triggered immunity in both host and non-host species. Overall, our study indicates that AbrNLPs are functionally and spatially (subcellular location) distinct and may play different but important roles during the pathogenesis of A. brassicae.

scholarly journals Functional characterisation of the Nep1-like protein effectors of the necrotrophic pathogen - Alternaria brassicae

2021 ◽  
Author(s):  
Deepak Duhan ◽  
Shivani Gajbhiye ◽  
Rajdeep Jaswal ◽  
Ravindra Pal Singh ◽  
Tilak Raj Sharma ◽  
...  
Keyword(s):  
Cell Death ◽  
Genome Sequence ◽  
Systematic Study ◽  
Subcellular Location ◽  
Ethylene Biosynthesis ◽  
Alternaria Brassicae ◽  
Necrotrophic Pathogen ◽  
Molecular Pattern ◽  
Functional Characterisation ◽  
Pathogen Associated Molecular Pattern

Alternaria brassicae is an important necrotrophic pathogen that infects the Brassicaceae family. A. brassicae, like other necrotrophs also secretes various proteinaceous effectors and metabolites that cause cell death to establish itself in the host. However, there has been no systematic study of A. brassicae effectors and their roles in pathogenesis. The availability of the genome sequence of A. brassicae has enabled the search for effectors and their functional characterisation. Nep1-like proteins are a superfamily of proteins that induce necrosis and ethylene biosynthesis. They have been reported from a variety of microbes including bacteria, fungi, and oomycetes. In this study, we identified two NLPs from A. brassicae viz. AbrNLP1 and AbrNLP2 and functionally characterised them. Although both AbrNLPs were found to be secretory in nature, they localised differentially inside the plant. AbrNLP2 was found to induce necrosis in both host and nonhost species, while AbrNLP1 could not induce necrosis in both species. Additionally, AbrNLP2 was shown to induce pathogen-associated molecular pattern (PAMP)-triggered immunity in both host and nonhost species. Overall, our study indicates that AbrNLPs are functionally and spatially (subcellular location) distinct and may play different but important roles during the pathogenesis of A. brassicae.

scholarly journals Leaping into the Unknown World of Sporisorium scitamineum Candidate Effectors

2020 ◽  
Vol 6 (4) ◽  
pp. 339
Author(s):  
Natália Sousa Teixeira-Silva ◽  
Patrícia Dayane Carvalho Schaker ◽  
Hugo Vianna Silva Rody ◽  
Thiago Maia ◽  
Christopher M. Garner ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Expression Profiles ◽  
Functional Characterization ◽  
Subcellular Location ◽  
Sporisorium Scitamineum ◽  
Effector Genes ◽  
Molecular Events ◽  
Phosphatase 2A ◽  
Set Up

Sporisorium scitamineum is a biotrophic fungus causing sugarcane smut disease. In this study, we set up a pipeline and used genomic and dual transcriptomic data previously obtained by our group to identify candidate effectors of S. scitamineum and their expression profiles in infected smut-resistant and susceptible sugarcane plants. The expression profile of different genes after infection in contrasting sugarcane genotypes assessed by RT-qPCR depended on the plant genotypes and disease progression. Three candidate effector genes expressed earlier only in resistant plants, four expressed in both genotypes, and three later in susceptible plants. Ten genes were cloned and transiently expressed in N. benthamiana leaves to determine their subcellular location, while four localized in more than one compartment. Two candidates, g3890 having a nucleoplasmic and mitochondrial location and g5159 targeting the plant cell wall, were selected to obtain their possible corresponding host targets using co-immunoprecipitation (CoIP) experiments and mass spectrometry. Various potential interactors were identified, including subunits of the protein phosphatase 2A and an endochitinase. We investigated the presence of orthologs in sugarcane and using transcriptome data present their expression profiles. Orthologs of sugarcane shared around 70% similarity. Identifying a set of putative fungal effectors and their plant targets provides a valuable resource for functional characterization of the molecular events leading to smut resistance in sugarcane plants and uncovers further opportunities for investigation.

Functional characterization of polypeptide release factor 1b in the ciliate Euplotes

2010 ◽  
Vol 30 (6) ◽  
pp. 425-431 ◽  
Author(s):  
Yan Wang ◽  
Baofeng Chai ◽  
Wei Wang ◽  
Aihua Liang
Keyword(s):  
Stop Codon ◽  
Functional Characterization ◽  
Subcellular Location ◽  
Class I ◽  
Release Factor ◽  
Amino Acid Motif ◽  
Codon Recognition ◽  
Stop Codon Recognition ◽  
Higher Eukaryotes

In higher eukaryotes, RF-I (class I release factor) [eRF1 (eukaryotic release factor 1)] is responsible for stop codon recognition and promotes nascent polypeptide release from the ribosome. Interestingly, two class I RFs, eRF1a and eRF1b, have been identified among the ciliates Euplotes, which are variant code organisms. In the present study, we analysed the comparative expression of eRF1a and eRF1b in Euplotes cells, demonstrating that the expression of eRF1b was higher than that of eRF1a. An interaction between eRF1b and eRF3 was confirmed, suggesting that an eRF1b function is facilitated by eRF3. Co-localization of both eRF1s indicated that they function in the same subcellular location in Euplotes cells. We also analysed the characteristics of stop codon discrimination by eRF1b. Like eRF1a, eRF1b recognized UAA and UAG as stop codons, but not UGA. This finding disagreed with the deduced characteristics of eRF1a/eRF1b from the classic hypothesis of ‘anticodon-mimicry’ proposed by Muramatsu et al. [Muramatsu, Heckmann, Kitanaka and Kuchino (2001) FEBS Lett. 488, 105–109]. Mutagenesis experiments indicated that the absolutely conserved amino acid motif ‘G31T32’ (numbered as for human eRF1) in eRF1b was the key to efficient stop codon recognition by eRF1b. In conclusion, these findings support and improve the ‘cavity model’ of stop codon discrimination by eRF1 proposed by Bertram et al. [Bertram, Bell, Ritchie, Fullerton and Stansfield (2000) RNA 6, 1236–1247] and Inagaki et al. [Inagaki, Blouin, Doolittle and Roger (2002) Nucleic Acids Res. 30, 532–544].

Niemann-Pick type C disease: Subcellular location and functional characterization of NPC2 proteins with naturally occurring missense mutations

2005 ◽  
Vol 26 (1) ◽  
pp. 20-28 ◽  
Author(s):  
Karim Chikh ◽  
Céline Rodriguez ◽  
Sébastien Vey ◽  
Marie T. Vanier ◽  
Gilles Millat
Keyword(s):  
Functional Characterization ◽  
Subcellular Location ◽  
Missense Mutations ◽  
Naturally Occurring ◽  
Type C ◽  
Niemann Pick

scholarly journals Identification of Nicotiana benthamiana Genes Involved in Pathogen-Associated Molecular Pattern–Triggered Immunity

2010 ◽  
Vol 23 (6) ◽  
pp. 715-726 ◽  
Author(s):  
Suma Chakravarthy ◽  
André C. Velásquez ◽  
Sophia K. Ekengren ◽  
Alan Collmer ◽  
Gregory B. Martin
Keyword(s):  
Cell Death ◽  
Nicotiana Benthamiana ◽  
Large Scale ◽  
Forward Genetics ◽  
Hormone Signaling ◽  
Virus Induced Gene Silencing ◽  
Molecular Pattern ◽  
Cell Death Response ◽  
A Cell ◽  
Pathogen Associated Molecular Pattern

In order to identify components of pathogen-associated molecular pattern–triggered immunity (PTI) pathways in Nicotiana benthamiana, we conducted a large-scale forward-genetics screen using virus-induced gene silencing and a cell-death-based assay for assessing PTI. The assay relied on four combinations of PTI-inducing nonpathogens and cell-death-causing challenger pathogens and was first validated in plants silenced for FLS2 or BAK1. Over 3,200 genes were screened and 14 genes were identified that, when silenced, compromised PTI as judged by the cell-death-based assay. Further analysis indicated that the 14 genes were not involved in a general cell death response. A subset of the genes was found to act downstream of FLS2-mediated PTI induction, and silencing of three genes compromised production of reactive oxygen species in leaves exposed to flg22. The 14 genes encode proteins with potential functions in defense and hormone signaling, protein stability and degradation, energy and secondary metabolism, and cell wall biosynthesis and provide a new resource to explore the molecular basis for the involvement of these processes in PTI.

scholarly journals Subtype identification and functional characterization of ryanodine receptors in rat cerebral artery myocytes

2010 ◽  
Vol 299 (2) ◽  
pp. C264-C278 ◽  
Author(s):  
Thirumalini Vaithianathan ◽  
Damodaran Narayanan ◽  
Maria T. Asuncion-Chin ◽  
Loice H. Jeyakumar ◽  
Jianxi Liu ◽  
...  
Keyword(s):  
Cerebral Artery ◽  
Ryanodine Receptors ◽  
Functional Characterization ◽  
Subcellular Location ◽  
Maximal Activity ◽  
Ruthenium Red ◽  
Voltage Curve ◽  
Subtype Identification ◽  
Steady State Activity

Ryanodine receptors (RyRs) regulate contractility in resistance-size cerebral artery smooth muscle, yet their molecular identity, subcellular location, and phenotype in this tissue remain unknown. Following rat resistance-size cerebral artery myocyte sarcoplasmic reticulum (SR) purification and incorporation into POPE-POPS-POPC (5:3:2; wt/wt) bilayers, unitary conductances of 110 ± 8, 334 ± 15, and 441 ± 27 pS in symmetric 300 mM Cs+ were usually detected. The most frequent (34/40 bilayers) conductance (334 pS) decreased to ≤100 pS when Cs+ was replaced with Ca2+. The predominant conductance displayed 66 bursts/min with at least three open and three closed states. The steady-state activity (NPo)-voltage curve was bell shaped, with NPo drastically decreasing when voltage was switched from −30 to −40 mV. NPo increased when intracellular calcium (Ca2+i) was raised within 0.1–100 μM to abruptly diminish with higher Ca2+i. Thus maximal activity occurred within the Ca2+i range found in rat cerebral artery myocytes under physiological conditions. NPo was reduced by ruthenium red (80 μM), increased monotonically by caffeine (0.1–5 mM) or ryanodine (0.05–5 μM), and unaffected by heparin (2 mg/ml). This phenotype resembles that of cardiac RyR and recombinant RyR2. RT-PCR detected RyR1, RyR2, and RyR3 transcripts in cerebral artery myocytes. However, real-time PCR indicated that RyR2 was 4 and 1.5 times more abundant than RyR1 and RyR3, respectively. Consistently, Western blotting showed that the RyR2 product was very abundant. Immunofluorescence showed that each RyR isoform distributed differentially among subcellular compartments. In particular, RyR2 was drastically stronger in the subplasmalemma than in other compartments, underscoring the predominance of RyR2 in a compartment where SR is abundant. Consistently, RyR from SR-enriched membranes displayed pharmacological specificity typical of RyR2, being activated by digoxin (1 μM), resistant to dantrolene (100 μM), and shifted to a subconductance by neomycin (100 nM). Therefore, RyR2 is the predominant molecular and functional RyR that is expressed in the SR membrane of rat resistance-size cerebral artery myocytes.

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