Accumulation of soluble proteins in the endoplasmic reticulum (ER) of plants is mediated by a receptor termed ER RETENTION DEFECTIVE2 (ERD2) or K/HDEL receptor. Using two gain-of-function assays and by complementing loss of function in , we discovered that compromising the lumenal N terminus or the cytosolic C terminus with fluorescent fusions abolishes its biological function and profoundly affects its subcellular localization. Based on the confirmed asymmetrical topology of ERD2, we engineered a new fluorescent ERD2 fusion protein that retains biological activity. Using this fusion, we show that ERD2 is exclusively detected at the Golgi apparatus, unlike nonfunctional C-terminal fusions, which also label the ER. Moreover, ERD2 is confined to early Golgi compartments and does not show ligand-induced redistribution to the ER. We show that the cytosolic C terminus of ERD2 plays a crucial role in its function. Two conserved leucine residues that do not correspond to any known targeting motifs for ER-Golgi trafficking were shown to be essential for both ERD2 Golgi residency and its ability to mediate ER retention of soluble ligands. The results suggest that anterograde ER to Golgi transport of ERD2 is either extremely fast, well in excess of the bulk flow rate, or that ERD2 does not recycle in the way originally proposed.
Summary Candidate effectors from lettuce downy mildew (Bremia lactucae) enable high‐throughput germplasm screening for the presence of resistance (R) genes. The nonhost species Lactuca saligna comprises a source of B. lactucae R genes that has hardly been exploited in lettuce breeding. Its cross‐compatibility with the host species L. sativa enables the study of inheritance of nonhost resistance (NHR).We performed transient expression of candidate RXLR effector genes from B. lactucae in a diverse Lactuca germplasm set. Responses to two candidate effectors (BLR31 and BLN08) were genetically mapped and tested for co‐segregation with disease resistance.BLN08 induced a hypersensitive response (HR) in 55% of the L. saligna accessions, but responsiveness did not co‐segregate with resistance to Bl:24. BLR31 triggered an HR in 5% of the L. saligna accessions, and revealed a novel R gene providing complete B. lactucae race Bl:24 resistance. Resistant hybrid plants that were BLR31 nonresponsive indicated other unlinked R genes and/or nonhost QTLs.We have identified a candidate avirulence effector of B. lactucae (BLR31) and its cognate R gene in L. saligna. Concurrently, our results suggest that R genes are not required for NHR of L. saligna.
Summary To cause disease in lettuce, the biotrophic oomycete Bremia lactucae secretes potential RxLR effector proteins. Here we report the discovery of an effector‐target hub consisting of four B. lactucae effectors and one lettuce protein target by a yeast‐two‐hybrid (Y2H) screening. Interaction of the lettuce tail‐anchored NAC transcription factor, LsNAC069, with B. lactucae effectors does not require the N‐terminal NAC domain but depends on the C‐terminal region including the transmembrane domain. Furthermore, in Y2H experiments, B. lactucae effectors interact with Arabidopsis and potato tail‐anchored NACs, suggesting that they are conserved effector targets. Transient expression of RxLR effector proteins BLR05 and BLR09 and their target LsNAC069 in planta revealed a predominant localization to the endoplasmic reticulum. Phytophthora capsici culture filtrate and polyethylene glycol treatment induced relocalization to the nucleus of a stabilized LsNAC069 protein, lacking the NAC‐domain (LsNAC069ΔNAC). Relocalization was significantly reduced in the presence of the Ser/Cys‐protease inhibitor TPCK indicating proteolytic cleavage of LsNAC069 allows for relocalization. Co‐expression of effectors with LsNAC069ΔNAC reduced its nuclear accumulation. Surprisingly, LsNAC069 silenced lettuce lines had decreased LsNAC069 transcript levels but did not show significantly altered susceptibility to B. lactucae. In contrast, LsNAC069 silencing increased resistance to Pseudomonas cichorii bacteria and reduced wilting effects under moderate drought stress, indicating a broad role of LsNAC069 in abiotic and biotic stress responses.
Summary Plant‐pathogenic oomycetes secrete effector proteins to suppress host immune responses. Resistance proteins may recognize effectors and activate immunity, which is often associated with a hypersensitive response (HR). Transient expression of effectors in plant germplasm and screening for HR has proven to be a powerful tool in the identification of new resistance genes. In this study, 14 effectors from the lettuce downy mildew Bremia lactucae race Bl:24 were screened for HR induction in over 150 lettuce accessions. Three effectors—BLN06, BLR38 and BLR40—were recognized in specific lettuce lines. The recognition of effector BLR38 in Lactuca serriola LS102 did not co‐segregate with resistance against race Bl:24, but was linked to resistance against multiple other B. lactucae races. Two unlinked loci are both required for effector recognition and are located near known major resistance clusters. Gene dosage affects the intensity of the BLR38‐triggered HR, but is of minor importance for disease resistance.
Microbial plant pathogens use secreted effector proteins for successful infection of their host. This evolved state is rather exceptional as most microbes do not cause disease in the vast majority of plant species. An important primary activity of effectors is to interfere with a range of plant immune processes to evade and suppress pathogen detection, or to block immune signalling and downstream responses. Furthermore, effectors can enhance disease susceptibility by altering cellular processes and modulating host transcription. For most of these activities, effectors specifically target plant proteins that are central in these processes. An advanced virulence strategy is the post‐translational modification by effectors of plant targets to change their activity or stability. The knowledge gathered on the molecular mechanisms underlying effector‐triggered susceptibility of plants provides great potential for novel approaches of resistance breeding. Key Concepts Pathogens secrete and/or translocate effector proteins to promote plant disease. Besides their primary role in promoting disease, effectors or effector‐modified plant proteins can be recognised by resistance proteins to activate an effector‐triggered immune response. Many effectors block pathogen‐associated molecular pattern (PAMP)‐triggered immunity and/or effector‐triggered immunity. Other effectors rewire signalling pathways and reprogramme the plant cell to promote pathogen growth. Certain effectors can affect the activity or function of host proteins by post‐translational modifications e.g. (de)phosphorylation or targeting for proteosomal degradation.
Plant 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 physical interactions between B. lactucae effectors and lettuce candidate target proteins. Using a lettuce cDNA library-based yeasttwo-hybrid system, 61 protein-protein interactions were identified, involving 21 B. lactucae effectors and 46 unique lettuce proteins. The top ten interactors based on the number of independent colonies identified in the Y2H and two interactors that belong to gene families involved in plant immunity, were further characterized. We determined the subcellular localization of the fluorescently tagged lettuce proteins and their interacting effectors. Importantly, relocalization of effectors or their interactors to the nucleus was observed for four proteinpairs upon their co-expression, supporting their interaction in planta.
21Plant pathogenic bacteria, fungi and oomycetes secrete effector proteins to manipulate host 22 cell processes to establish a successful infection. Over the last decade the genomes and transcriptomes 23 of many agriculturally important plant pathogens have been sequenced and vast candidate effector 24 repertoires were identified using bioinformatic analyses. Elucidating the contribution of individual 25 effectors to pathogenicity is the next major hurdle. To advance our understanding of the molecular 26 mechanisms underlying lettuce susceptibility to the downy mildew Bremia lactucae, we mapped a 27 network of physical interactions between B. lactucae effectors and lettuce target proteins. Using a 28 lettuce cDNA library-based yeast-two-hybrid system, 61 protein-protein interactions were identified, 29 involving 21 B. lactucae effectors and 46 unique lettuce proteins. The top ten targets based on the 30 number of independent colonies identified in the Y2H and two targets that belong to gene families 31 involved in plant immunity, were further characterized. We determined the subcellular localization of 32 the fluorescently tagged target proteins and their interacting effectors. Importantly, relocalization of 33 effectors or targets to the nucleus was observed for four effector-target pairs upon their co-expression, 34 supporting their interaction in planta. 42 nucleotide-binding and leucine-rich repeat receptors (NLRs) recognize host translocated effectors, or 43 the perturbations effectors induce on host proteins, resulting in the activation of effector-triggered 44 immunity (ETI). In turn, ETI can be counteracted by other effectors leading to a state of effector-45 triggered susceptibility (ETS) [1]. 46 Fungi and oomycetes secrete apoplastic effectors that operate at the host-pathogen interface, 47 and host-translocated effectors that act intracellularly in the host. The genomes of different pathogens 48 encode for extensive candidate effector sets, which have specific characteristics based on their origin. 49 Fungal genomes encode e.g. small apoplastic cysteine-rich proteins [2], plant pathogenic downy 50 mildews and Phytophthora species express host-translocated Crinklers and RXLR effectors [3-6] 51 whereas plant pathogenic Gram-negative bacteria, e.g. Pseudomonas syringae, inject type III effectors 52 into host cells [7]. At present, the major challenge lies in elucidating the contribution of individual 53 effectors to the infection process through the identification of plant targets and analysis of the 54 molecular mechanisms that contribute to disease susceptibility. 55 Effector targets were systematically studied in Arabidopsis thaliana by identifying physical 56 interactions between Arabidopsis proteins and effector proteins of the bacterium P. syringae, the 57 obligate biotrophic oomycete Hyaloperonospora arabidopsidis, and the obligate biotrophic 58 ascomycete Golovinomyces orontii resulting from a yeast-two-hybrid (Y2H) screening of ~8000 59 Arabidopsis ORFs [8,9]. Interactions between 123 effectors a...
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