Leaping into the Unknown World of Sporisorium scitamineum Candidate Effectors

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.

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Visual Biochemistry: modular microfluidics enables kinetic insight from time-resolved cryo-EM

10.1101/2020.03.04.972604 ◽

2020 ◽

Cited By ~ 1

Author(s):

Märt-Erik Mäeots ◽

Byungjin Lee ◽

Andrea Nans ◽

Seung-Geun Jeong ◽

Mohammad M. N. Esfahani ◽

...

Keyword(s):

Sample Preparation ◽

Reaction Intermediates ◽

Protein Distribution ◽

Time Resolved ◽

Biochemical Processes ◽

Molecular Events ◽

Biophysical Analysis ◽

Common Strategy ◽

Set Up

AbstractMechanistic understanding of biochemical reactions requires structural and kinetic characterization of the underlying chemical processes. However, no single experimental technique can provide this information in a broadly applicable manner and thus structural studies of static macromolecules are often complemented by biophysical analysis. Moreover, the common strategy of utilizing mutants or crosslinking probes to stabilize otherwise short-lived reaction intermediates is prone to trapping off-pathway artefacts and precludes determining the order of molecular events. To overcome these limitations and allow visualisation of biochemical processes at near-atomic spatial resolution and millisecond time scales, we developed a time-resolved sample preparation method for cryo-electron microscopy (trEM). We integrated a modular microfluidic device, featuring a 3D-mixing unit and a delay line of variable length, with a gas-assisted nozzle and motorised plunge-freeze set-up that enables automated, fast, and blot-free sample vitrification. This sample preparation not only preserves high-resolution structural detail but also substantially improves protein distribution across the vitreous ice. We validated the method by examining the formation of RecA filaments on single-stranded DNA. We could reliably visualise reaction intermediates of early filament growth across three orders of magnitude on sub-second timescales. Quantification of the trEM data allowed us to characterize the kinetics of RecA filament growth. The trEM method reported here is versatile, easy to reproduce and thus readily adaptable to a broad spectrum of fundamental questions in biology.

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Functional characterization of genes mediating cell wall metabolism and responses to plant cell wall integrity impairment

BMC Plant Biology ◽

10.1186/s12870-019-1934-4 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 3

Author(s):

Timo Engelsdorf ◽

Lars Kjaer ◽

Nora Gigli-Bisceglia ◽

Lauri Vaahtera ◽

Stefan Bauer ◽

...

Keyword(s):

Cell Wall ◽

Plant Cell ◽

Plant Cell Wall ◽

Functional Characterization ◽

Cell Wall Integrity ◽

Cell Wall Metabolism

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Expression Profiles Analysis and Functional Characterization of MicroRNA-660 in Skeletal Muscle Differentiation

Journal of Cellular Biochemistry ◽

10.1002/jcb.25901 ◽

2017 ◽

Vol 118 (8) ◽

pp. 2387-2394 ◽

Cited By ~ 3

Author(s):

Binglin Yue ◽

Jiyao Wu ◽

Yanhuan Wang ◽

Chunlei Zhang ◽

Xingtang Fang ◽

...

Keyword(s):

Skeletal Muscle ◽

Expression Profiles ◽

Functional Characterization ◽

Muscle Differentiation ◽

Skeletal Muscle Differentiation

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Correction to: Functional characterization of genes mediating cell wall metabolism and responses to plant cell wall integrity impairment

BMC Plant Biology ◽

10.1186/s12870-019-1995-4 ◽

2019 ◽

Vol 19 (1) ◽

Author(s):

Timo Engelsdorf ◽

Lars Kjaer ◽

Nora Gigli-Bisceglia ◽

Lauri Vaahtera ◽

Stefan Bauer ◽

...

Keyword(s):

Cell Wall ◽

Plant Cell ◽

Plant Cell Wall ◽

Functional Characterization ◽

Cell Wall Integrity ◽

Cell Wall Metabolism

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

Bioscience Reports ◽

10.1042/bsr20090154 ◽

2010 ◽

Vol 30 (6) ◽

pp. 425-431 ◽

Cited By ~ 9

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].

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Expression Profiles and Functional Characterization of Two Odorant-Binding Proteins From the Apple Buprestid Beetle Agrilus mali (Coleoptera: Buprestidae)

Journal of Economic Entomology ◽

10.1093/jee/toy066 ◽

2018 ◽

Vol 111 (3) ◽

pp. 1420-1432 ◽

Cited By ~ 12

Author(s):

Xiaoning Cui ◽

Deguang Liu ◽

Keke Sun ◽

Yang He ◽

Xiaoqin Shi

Keyword(s):

Binding Proteins ◽

Expression Profiles ◽

Functional Characterization ◽

Odorant Binding Proteins ◽

Odorant Binding

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Key wheat GRF genes constraining wheat tillering of mutant dmc

PeerJ ◽

10.7717/peerj.11235 ◽

2021 ◽

Vol 9 ◽

pp. e11235

Author(s):

Jing Zhang ◽

Junchang Li ◽

Yongjing Ni ◽

Yumei Jiang ◽

Zhixin Jiao ◽

...

Keyword(s):

Triticum Aestivum ◽

Acetic Acid ◽

Indole Acetic Acid ◽

Expression Profiles ◽

Functional Characterization ◽

Tissue Expression ◽

Wild Type ◽

Gene Structures ◽

Wheat Mutant

Tillering is a key agronomy trait for wheat (Triticum aestivum L.) production. Previously, we have reported a dwarf-monoculm wheat mutant (dmc) obtained from cultivar Guomai 301 (wild type, WT), and found growth regulating factors (GRFs) playing important roles in regulating wheat tillering. This study is to systematically investigate the roles of all the wheat GRFs (T. aestivum GRFs, TaGRFs) in regulating tillering, and screen out the key regulators. A total of 30 TaGRFs were identified and their physicochemical properties, gene structures, conserved domains, phylogenetic relationships and tissue expression profiles were analyzed. The expression levels of all the TaGRFs were significantly lower in dmc than those in WT at early tillering stage, and the abnormal expressions of TaGRF2-7(A, B, D), TaGRF5-7D, TaGRF10-6(A, B, D) and TaGRF11-2A were major causes constraining the tillering of dmc. The transcriptions of TaGRFs were significantly affected by exogenous indole acetic acid (IAA) and gibberellin acid (GA3) applications, which suggested that TaGRFs as well as IAA, GA signaling were involved in controlling wheat tillering. This study provided valuable clues for functional characterization of GRF genes in wheat.

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GENOME-WIDE IDENTIFICATION AND COMPUTATIONAL CHARACTERIZATION OF THE NUCLEAR FACTOR-YC SUB-UNITS IN GRAIN AMARANTH (Amaranthus hypochondriacus)

Journal of Science Natural Science ◽

10.18173/2354-1059.2021-0057 ◽

2021 ◽

Vol 66 (3) ◽

pp. 161-169

Author(s):

Huyen Tran Thi Thanh ◽

Hong La Viet ◽

Quynh Le Thi Ngoc ◽

Thuy Pham Chau ◽

Quyen Ha Thi ◽

...

Keyword(s):

Growth And Development ◽

Expression Profiles ◽

Functional Characterization ◽

Chemical Properties ◽

Nuclear Factor ◽

Grain Amaranth ◽

Amaranthus Hypochondriacus ◽

Grand Average ◽

Genes Encoding

Nuclear factor-Y (NF-Y) has been known as one of the plant-specific transcription factors that play key roles in numerous biological processes during the growth and development of plant species. In this study, a comprehensive analysis of NF-YC sub-units in grain amaranth (Amaranthus hypochondriacus) was carried out based on the bioinformatics approaches. Firstly, a total of five members of the NF-YC sub-units was reported in the grain amaranth. Its structural analyses revealed that the NF-YC sub-units were variable in physic-chemical properties, like protein sizes, molecular masses, isoelectric point, instability index, and grand average of hydropathy. Of our interest, the expression profiles of genes encoding NF-YC sub-units in various tissues\organs during the growth and development of grain amaranth. We found that three genes, including AhNF-YC01, AhNF-YC04, and AhNF-YC05 were highly expressed in leaf, root, floral, immature seed, and stem tissues. Interestingly, AhNF-YC05 was exclusively expressed in leaf and stem tissues. Taken together, our study could provide a solid understanding for further functional characterization of genes encoding NF-YC sub-units in grain amaranth.

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Expression Characteristics and Functional Analysis of the ScWRKY3 Gene from Sugarcane

International Journal of Molecular Sciences ◽

10.3390/ijms19124059 ◽

2018 ◽

Vol 19 (12) ◽

pp. 4059 ◽

Cited By ~ 5

Author(s):

Ling Wang ◽

Feng Liu ◽

Xu Zhang ◽

Wenju Wang ◽

Tingting Sun ◽

...

Keyword(s):

Transcriptional Activation ◽

Functional Characterization ◽

Bacterial Pathogen ◽

Future Research ◽

Sporisorium Scitamineum ◽

Transcriptional Regulatory ◽

Transient Overexpression ◽

Hybrid Cultivar ◽

Sugarcane Stem

The plant-specific WRKY transcriptional regulatory factors have been proven to play vital roles in plant growth, development, and responses to biotic and abiotic stresses. However, there are few studies on the WRKY gene family in sugarcane (Saccharum spp.). In the present study, the characterization of a new subgroup, IIc WRKY protein ScWRKY3, from a Saccharum hybrid cultivar is reported. The ScWRKY3 protein was localized in the nucleus of Nicotiana benthamiana leaves and showed no transcriptional activation activity and no toxic effects on the yeast strain Y2HGold. An interaction between ScWRKY3 and a reported sugarcane protein ScWRKY4, was confirmed in the nucleus. The ScWRKY3 gene had the highest expression level in sugarcane stem pith. The transcript of ScWRKY3 was stable in the smut-resistant Saccharum hybrid cultivar Yacheng05-179, while it was down-regulated in the smut-susceptible Saccharum hybrid cultivar ROC22 during inoculation with the smut pathogen (Sporisorium scitamineum) at 0–72 h. ScWRKY3 was remarkably up-regulated by sodium chloride (NaCl), polyethylene glycol (PEG), and plant hormone abscisic acid (ABA), but it was down-regulated by salicylic acid (SA) and methyl jasmonate (MeJA). Moreover, transient overexpression of the ScWRKY3 gene in N. benthamiana indicated a negative regulation during challenges with the fungal pathogen Fusarium solani var. coeruleum or the bacterial pathogen Ralstonia solanacearum in N. benthamiana. The findings of the present study should accelerate future research on the identification and functional characterization of the WRKY family in sugarcane.

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