scholarly journals Host targets of effectors of the lettuce downy mildew Bremia lactucae from cDNA-based yeast two-hybrid screening

2019 ◽  
Author(s):  
Alexandra J.E. Pelgrom ◽  
Claudia-Nicole Meisrimler ◽  
Joyce Elberse ◽  
Thijs Koorman ◽  
Mike Boxem ◽  
...  
Keyword(s):  
Downy Mildew ◽  
Pathogenic Bacteria ◽  
Plant Pathogens ◽  
Molecular Mechanisms ◽  
Effector Proteins ◽  
Bremia Lactucae ◽  
Yeast Two Hybrid ◽  
Target Proteins ◽  
Successful Infection ◽  
Two Hybrid

AbstractPlant pathogenic bacteria, fungi and oomycetes secrete effector proteins to manipulate host cell processes to establish a successful infection. Over the last decade the genomes and transcriptomes of many agriculturally important plant pathogens have been sequenced and vast candidate effector repertoires were identified using bioinformatic analyses. Elucidating the contribution of individual effectors to pathogenicity is the next major hurdle. To advance our understanding of the molecular mechanisms underlying lettuce susceptibility to the downy mildew Bremia lactucae, we mapped a network of physical interactions between B. lactucae effectors and lettuce target proteins. Using a lettuce cDNA library-based yeast-two-hybrid system, 61 protein-protein interactions were identified, involving 21 B. lactucae effectors and 46 unique lettuce proteins. The top ten targets based on the number of independent colonies identified in the Y2H and two targets that belong to gene families involved in plant immunity, were further characterized. We determined the subcellular localization of the fluorescently tagged target proteins and their interacting effectors. Importantly, relocalization of effectors or targets to the nucleus was observed for four effector-target pairs upon their co-expression, supporting their interaction in planta.

scholarly journals Host interactors of effector proteins of the lettuce downy mildew Bremia lactucae obtained by yeast two-hybrid screening

PLoS ONE ◽  
2020 ◽  
Vol 15 (5) ◽  
pp. e0226540 ◽  
Author(s):  
Alexandra J. E. Pelgrom ◽  
Claudia-Nicole Meisrimler ◽  
Joyce Elberse ◽  
Thijs Koorman ◽  
Mike Boxem ◽  
...  
Keyword(s):  
Downy Mildew ◽  
Effector Proteins ◽  
Bremia Lactucae ◽  
Yeast Two Hybrid ◽  
Lettuce Downy Mildew ◽  
Two Hybrid ◽  
Hybrid Screening

scholarly journals YopJ Family Effectors Promote Bacterial Infection through a Unique Acetyltransferase Activity

2016 ◽  
Vol 80 (4) ◽  
pp. 1011-1027 ◽  
Author(s):  
Ka-Wai Ma ◽  
Wenbo Ma
Keyword(s):  
Plant Pathogens ◽  
Molecular Mechanisms ◽  
Bacterial Species ◽  
Cysteine Proteases ◽  
Catalytic Triad ◽  
Effector Proteins ◽  
Host Cells ◽  
Target Proteins ◽  
Acetyltransferase Activity ◽  
Type Iii

SUMMARYGram-negative bacterial pathogens rely on the type III secretion system to inject virulence proteins into host cells. These type III secreted “effector” proteins directly manipulate cellular processes to cause disease. Although the effector repertoires in different bacterial species are highly variable, theYersiniaouter protein J (YopJ) effector family is unique in that its members are produced by diverse animal and plant pathogens as well as a nonpathogenic microsymbiont. All YopJ family effectors share a conserved catalytic triad that is identical to that of the C55 family of cysteine proteases. However, an accumulating body of evidence demonstrates that many YopJ effectors modify their target proteins in hosts by acetylating specific serine, threonine, and/or lysine residues. This unique acetyltransferase activity allows the YopJ family effectors to affect the function and/or stability of their targets, thereby dampening innate immunity. Here, we summarize the current understanding of this prevalent and evolutionarily conserved type III effector family by describing their enzymatic activities and virulence functions in animals and plants. In particular, the molecular mechanisms by which representative YopJ family effectors subvert host immunity through posttranslational modification of their target proteins are discussed.

scholarly journals Implication of the Whitefly Protein Vps Twenty Associated 1 (Vta1) in the Transmission of Cotton Leaf Curl Multan Virus

2021 ◽  
Vol 9 (2) ◽  
pp. 304
Author(s):  
Yao Chi ◽  
Li-Long Pan ◽  
Shu-Sheng Liu ◽  
Shahid Mansoor ◽  
Xiao-Wei Wang
Keyword(s):  
Coat Protein ◽  
Molecular Mechanisms ◽  
Cotton Leaf ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Vacuolar Protein ◽  
Leaf Curl Disease ◽  
Two Hybrid ◽  
Asia Ii 1 ◽  
Leaf Curl

Cotton leaf curl Multan virus (CLCuMuV) is one of the major casual agents of cotton leaf curl disease. Previous studies show that two indigenous whitefly species of the Bemisia tabaci complex, Asia II 1 and Asia II 7, are able to transmit CLCuMuV, but the molecular mechanisms underlying the transmission are poorly known. In this study, we attempted to identify the whitefly proteins involved in CLCuMuV transmission. First, using a yeast two-hybrid system, we identified 54 candidate proteins of Asia II 1 that putatively can interact with the coat protein of CLCuMuV. Second, we examined interactions between the CLCuMuV coat protein and several whitefly proteins, including vacuolar protein sorting-associated protein (Vps) twenty associated 1 (Vta1). Third, using RNA interference, we found that Vta1 positively regulated CLCuMuV acquisition and transmission by the Asia II 1 whitefly. In addition, we showed that the interaction between the CLCuMuV coat protein and Vta1 from the whitefly Middle East-Asia Minor (MEAM1), a poor vector of CLCuMuV, was much weaker than that between Asia II 1 Vta1 and the CLCuMuV coat protein. Silencing of Vta1 in MEAM1 did not affect the quantity of CLCuMuV acquired by the whitefly. Taken together, our results suggest that Vta1 may play an important role in the transmission of CLCuMuV by the whitefly.

The role of oomycete effectors in plant - pathogen interactions

2010 ◽  
Vol 37 (10) ◽  
pp. 919 ◽  
Author(s):  
Adrienne R. Hardham ◽  
David M. Cahill
Keyword(s):  
Plant Cell ◽  
Plant Pathogens ◽  
Plant Defence ◽  
Effector Proteins ◽  
Plant Structure ◽  
Cell Wall Degrading Enzymes ◽  
Cell Cytoplasm ◽  
Diverse Range ◽  
Successful Infection ◽  
Pathogen Effector

Plants constantly come into contact with a diverse range of microorganisms that are potential pathogens, and they have evolved multi-faceted physical and chemical strategies to inhibit pathogen ingress and establishment of disease. Microbes, however, have developed their own strategies to counteract plant defence responses. Recent research on plant–microbe interactions has revealed that an important part of the infection strategies of a diverse range of plant pathogens, including bacteria, fungi and oomycetes, is the production of effector proteins that are secreted by the pathogen and that promote successful infection by manipulating plant structure and metabolism, including interference in plant defence mechanisms. Pathogen effector proteins may function either in the extracellular spaces within plant tissues or within the plant cell cytoplasm. Extracellular effectors include cell wall degrading enzymes and inhibitors of plant enzymes that attack invading pathogens. Intracellular effectors move into the plant cell cytoplasm by as yet unknown mechanisms where, in incompatible interactions, they may be recognised by plant resistance proteins but where, in compatible interactions, they may suppress the plant’s immune response. This article presents a brief overview of our current understanding of the nature and function of effectors produced by oomycete plant pathogens.

The Arabidopsis thaliana papain-like cysteine protease RD21 interacts with a root-knot nematode effector protein

Nematology ◽  
2015 ◽  
Vol 17 (6) ◽  
pp. 655-666 ◽  
Author(s):  
Laura J. Davies ◽  
Lei Zhang ◽  
Axel A. Elling
Keyword(s):  
Arabidopsis Thaliana ◽  
Cysteine Protease ◽  
Host Plants ◽  
Effector Proteins ◽  
Effector Protein ◽  
Yeast Two Hybrid ◽  
Root Knot Nematode ◽  
Second Stage ◽  
Stage Juvenile ◽  
Two Hybrid

The root-knot nematode Meloidogyne chitwoodi secretes effector proteins into the cells of host plants to manipulate plant-derived processes in order to achieve successful parasitism. Mc1194 is a M. chitwoodi effector that is highly expressed in pre-parasitic second-stage juvenile nematodes. Yeast two-hybrid assays revealed Mc1194 specifically interacts with a papain-like cysteine protease (PLCP), RD21A in Arabidopsis thaliana. Mc1194 interacts with both the protease and granulin domains of RD21A. PLCPs are targeted by effectors secreted by bacterial, fungal and oomycete pathogens and the hypersusceptibility of rd21-1 mutants to M. chitwoodi indicates RD21A plays a role in plant-parasitic nematode infection.

scholarly journals Interactions of Target Proteins with the virB4 Gene from Bovine Embryo Trophoblast Cells Infected with Brucella abortus Screened Through a Yeast Two-Hybrid Assay

2012 ◽  
Vol 11 (11) ◽  
pp. 1793-1799
Author(s):  
Chen Chuangfu ◽  
Zhang Hui ◽  
Wang Pengyan ◽  
Wang Yuanzhi ◽  
Guo Qian ◽  
...  
Keyword(s):  
Brucella Abortus ◽  
Bovine Embryo ◽  
Trophoblast Cells ◽  
Yeast Two Hybrid ◽  
Target Proteins ◽  
Two Hybrid

scholarly journals Identifying Interacting Proteins of Arabidopsis Cyclophilin38 (AtCYP38), a Key Factor for PSII Assembly/Repair, via Multiple Screening Approaches

2020 ◽  
Author(s):  
Yaqi Hao ◽  
Jiashu Chu ◽  
Lujing Shi ◽  
Cong Ma ◽  
Liangliang Hui ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Thylakoid Membrane ◽  
Interacting Proteins ◽  
Yeast Two Hybrid ◽  
Working Mechanism ◽  
Web Based ◽  
Thylakoid Lumen ◽  
Target Proteins ◽  
Thylakoid Membrane Proteins ◽  
Two Hybrid

Abstract BackgroundAtCYP38, a thylakoid lumen localized immunophilin, is essential for photosystem II (PSII) assembly and maintenance, but how AtCYP38 functions in chloroplast remains unknown. Based on previous functional studies and its crystal structure, we hypothesize that AtCYP38 should function via binding its targets or cofactors in the thylakoid lumen to influence PSII performance. Therefore, identifying its target proteins and cofactors would be a key step to understand the working mechanism of AtCYP38.ResultsTo identify potential interacting proteins of AtCYP38, we first adopted two web-based tools, ATTED-II and STRING, and found 15 proteins functionally related to AtCYP38. We then screened a yeast two-hybrid library including an Arabidopsis genome wide cDNA with the N-terminal domain, the C-terminal domain, and the full-length mature protein of AtCYP38. 25 positive targets were identified, but a very limited number of target proteins were localized in the thylakoid lumen. In order to specifically search interacting proteins of AtCYP38 in the thylakoid lumen, we created a yeast two-hybrid mini library including the thylakoid lumenal proteins and lumen fractions of thylakoid membrane proteins. After screening the mini library with 3 different forms of AtCYP38, we obtained 6 thylakoid membrane proteins and 9 thylakoid lumenal proteins as interacting proteins of AtCYP38. We further confirmed the localization of several identified proteins and their interaction between AtCYP38.ConclusionsAfter analysis with two web-based tools and yeast two-hybrid screenings against two different libraries, we identified a couple of potential interacting proteins, which could be functionally related to AtCYP38. We believe that the results will lay a foundation for unveiling the working mechanism of AtCYP38 in photosynthesis.

Type III secretion chaperones ShcS1 and ShcO1 from Pseudomonas syringae pv. tomato DC3000 bind more than one effector

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 269-280 ◽  
Author(s):  
Ute Kabisch ◽  
Angelika Landgraf ◽  
Jana Krause ◽  
Ulla Bonas ◽  
Jens Boch
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Gel Filtration ◽  
Effector Proteins ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
Tomato Dc3000 ◽  
Type Iii ◽  
Effector Genes ◽  
Two Hybrid

The hrp-type III secretion (TTS) system is a key pathogenicity factor of the plant pathogen Pseudomonas syringae pv. tomato DC3000 that translocates effector proteins into the cytosol of the eukaryotic host cell. The translocation of a subset of effectors is dependent on specific chaperones. In this study an operon encoding a TTS chaperone (ShcS1) and the truncated effector HopS1′ was characterized. Yeast two-hybrid analysis and pull-down assays demonstrated that these proteins interact. Using protein fusions to AvrRpt2 it was shown that ShcS1 facilitates the translocation of HopS1′, suggesting that ShcS1 is a TTS chaperone for HopS1′ and that amino acids 1 to 118 of HopS1′ are required for translocation. P. syringae pv. tomato DC3000 carries two shcS1 homologues, shcO1 and shcS2, which are located in different operons, and both operons include additional putative effector genes. Transcomplementation experiments showed that ShcS1 and ShcO1, but not ShcS2, can facilitate the translocation of HopS1′ : : AvrRpt2. To characterize the specificities of the putative chaperones, yeast two-hybrid interaction studies were performed between the three chaperones and putative target effectors. These experiments showed that both ShcS1 and ShcO1 bind to two different effectors, HopS1′ and HopO1-1, that share only 16 % amino acid sequence identity. Using gel filtration it was shown that ShcS1 forms homodimers, and this was confirmed by yeast two-hybrid experiments. In addition, ShcS1 is also able to form heterodimers with ShcO1. These data demonstrate that ShcS1 and ShcO1 are exceptional class IA TTS chaperones because they can bind more than one target effector.

scholarly journals Molecular mechanisms of host cell manipulation by plant pathogens

2014 ◽  
Vol 70 (a1) ◽  
pp. C801-C801
Author(s):  
Richard Hughes ◽  
Stuart King ◽  
Abbas Maqbool ◽  
Hazel McLellan ◽  
Tolga Bozkurt ◽  
...  
Keyword(s):  
Cell Death ◽  
Crop Production ◽  
Plant Pathogens ◽  
Molecular Mechanisms ◽  
Complex Structure ◽  
Effector Proteins ◽  
Plant Diseases ◽  
Cell Manipulation ◽  
Cell Physiology ◽  
Structural Studies

An estimated 15% of global crop production is lost to pre-harvest disease every year. New ways to manage plant diseases are required. A mechanistic understanding of how plant pathogens re-program their hosts to enable colonisation may provide novel genetic or chemical opportunities to interfere with disease. One notorious plant parasite is the Irish potato famine pathogen Phytophthora infestans. This pathogen remains a considerable threat to potato/tomato crops today as the agent of late blight. Plant pathogens secrete effector proteins outside of and into plant cells to suppress host defences and manipulate cell physiology. Structural studies have provided insights into effector evolution and enabled experiments to probe function [1-3]. Crystal structures of 4 Phytophthora RXLR-type effectors, which are unrelated in primary sequence, revealed similarities in the fold of these proteins. This fold was proposed to act as a stable scaffold that supports diversification of effectors. Further, molecular modelling has enabled mapping of single-site variants responsible for specialisation of a Phytophthora Cystatin-like effector, revealing how effectors can adapt to new hosts after a "host jump". Structural studies describing how RXLR-effectors interact with host targets are lacking. We have used Y2H/co-IP studies to identify host proteins that interact with P. infestans effectors PexRD2 and PexRD54. PexRD2 interacts with MAPKKKe, a component of plant immune signalling pathways, and suppressed cell death activities of this protein. We used the structure of PexRD2 to design mutants that fail to interact with MAPKKKe, and no longer suppress cell-death activities. We found that PexRD54 interacts with potato homologues of the autophagy protein ATG8. We have obtained a crystal structure for PexRD54 in the presence of ATG8. We are now using X-ray scattering to verify the complex structure in solution prior to establishing the role of this interaction during infection.

scholarly journals Specific In Planta Recognition of Two GKLR Proteins of the Downy Mildew Bremia lactucae Revealed in a Large Effector Screen in Lettuce

2013 ◽  
Vol 26 (11) ◽  
pp. 1259-1270 ◽  
Author(s):  
Joost H. M. Stassen ◽  
Erik den Boer ◽  
Pim W. J. Vergeer ◽  
Annemiek Andel ◽  
Ursula Ellendorff ◽  
...  
Keyword(s):  
Immune Responses ◽  
Downy Mildew ◽  
Hypersensitive Response ◽  
Effector Proteins ◽  
Chromosome 9 ◽  
Downy Mildew Resistance ◽  
Chromosome 2 ◽  
Bremia Lactucae ◽  
In Planta ◽  
Effector Triggered Immunity

Breeding lettuce (Lactuca sativa) for resistance to the downy mildew pathogen Bremia lactucae is mainly achieved by introgression of dominant downy mildew resistance (Dm) genes. New Bremia races quickly render Dm genes ineffective, possibly by mutation of recognized host-translocated effectors or by suppression of effector-triggered immunity. We have previously identified 34 potential RXLR(-like) effector proteins of B. lactucae that were here tested for specific recognition within a collection of 129 B. lactucae-resistant Lactuca lines. Two effectors triggered a hypersensitive response: BLG01 in 52 lines, predominantly L. saligna, and BLG03 in two L. sativa lines containing Dm2 resistance. The N-terminal sequences of BLG01 and BLG03, containing the signal peptide and GKLR variant of the RXLR translocation motif, are not required for in planta recognition but function in effector delivery. The locus responsible for BLG01 recognition maps to the bottom of lettuce chromosome 9, whereas recognition of BLG03 maps in the RGC2 cluster on chromosome 2. Lactuca lines that recognize the BLG effectors are not resistant to Bremia isolate Bl:24 that expresses both BLG genes, suggesting that Bl:24 can suppress the triggered immune responses. In contrast, lettuce segregants displaying Dm2-mediated resistance to Bremia isolate Bl:5 are responsive to BLG03, suggesting that BLG03 is a candidate Avr2 protein.

Sign in / Sign up

Export Citation Format

Share Document