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

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 yeast-two-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 protein-pairs upon their co-expression, supporting their interaction in planta.


Introduction
Plant pathogenic bacteria, fungi and oomycetes deploy effector proteins to manipulate host cell processes. Importantly, effectors serve to suppress and circumvent plant immune responses. Basal host defense responses are activated upon recognition of ubiquitously present microbe-associated molecular patterns (MAMPs) by plant pattern recognition receptors (PRRs). Evolutionary adapted pathogens deploy effectors to suppress this pattern-triggered immunity (PTI). Specialized intracellular nucleotide-binding and leucine-rich repeat receptors (NLRs) recognize host translocated effectors, or the perturbations effectors induce on host seedlings treated with the salicylic acid (SA) analog benzothiadiazole (BTH; 0.1 mg/ml) 24h after spraying. RNA originating from the different treatments, was mixed in equal amounts. A three-frame uncut oligo(dT)-primed cDNA library in pENTR222 was created from 2 mg RNA using Gateway cloning technology. The library was transferred into the yeast-two-hybrid destination vector pDEST22 to generate GAL4 activation domain (AD) lettuce fusion proteins. The pDEST22 library in E. coli strain DH10B originated from 22 x 10 6 colony forming units (cfu) with an average insert size of 1.1 kb. B. lactucae effectors were amplified using cDNA from the sequence encoding the predicted signal peptide cleavage sites and new start codons were introduced. Gateway entry clones of B. lactucae effectors were recombined with the pDEST32 yeast-two-hybrid destination vector using LR clonase to generate GAL4 DNA binding domain (DBD) effector fusion proteins.

Library screening
The mating method was used for library screening [14]. Diploid yeast cells were plated on Sc-Leu-Trp-His + amp medium to identify interacting bait-prey combinations. Retransformation of bait and prey candidates was performed to confirm interactions. A detailed description can be found in S1 File.

Bioinformatic analysis of effectors and lettuce interactors
Lettuce prey gene models were extracted using a BlastN search against the lettuce genome [15]. Where necessary, incomplete gene models were corrected using the lettuce transcriptome [16] and prey sequencing data. Prey sequences with a stop codon within the first 50 amino acids (aa) after the GAL4-AD sequence were considered not in frame. Presence of signal peptides was predicted using SignalP4.1 [17] and transmembrane domains were identified with TMHMM [18,19] using the default parameters. Transmembrane domains identified in effector sequences were validated using TOPCONS [20,21]. Domain prediction was performed using InterProScan5. The presence of importin-α dependent nuclear localization signals was predicted using cNLS Mapper [22]. WY domains were predicted using an HMM model [23] with an individual cut-off E-value of 0.001 for the best motif within an effector. Post-translational lipid modifications were predicted using GPS-Lipid/CSS-Palm [24,25] on effector sequences lacking the signal peptide.
L. sativa cv. Salinas coding sequences corresponding to genome v8 (Genome ID 28333) were downloaded from the CoGe platform (https://genomevolution.org/). Translated CDSs of interactors on the shortlist were searched using Hidden Markov Models for the presence of domains with E-value 1e -4 as the cut-off. In general, all gene models with the same Pfam domain were subsequently extracted to identify the corresponding gene families. All gene models belonging to the same gene family as the interactors on the shortlist were named collectively. Gene names are composed of the prefix 'Ls' for L. sativa followed by a short abbreviation denoting the domain and a number. Genes were numbered according to their position on the lettuce linkage groups 1-9 to avoid issues due to the, often, complex orthologous relationship with Arabidopsis proteins. An overview of all the new nomenclature for gene models can be found in S2 File. A detailed description of the procedure per gene model can be found in S1 File.

Transient expression in N. benthamiana
Full-length prey sequences were amplified from lettuce cv. Olof cDNA or prey plasmid using primers listed in S1 Table and recombined in a modified pGemTEasy vector containing the pDONR201 Gateway recombination site (pGemTEasy mod ) using BP clonase. Bait and prey entry clones were further recombined in pUBN-YFP-DEST and pUBN-CFP-DEST (kind gift from Dr. Christopher Grefen; [26]) or with pB7WGY2 and pB7WGC2 [27] to create N-terminal YFP and CFP fusion proteins respectively using LR clonase and transformed in Agrobacterium tumefaciens strain C58C1 (pGV2260) with selection on rifampicin (50 μg/ml), carbenicillin (50 μg/ml) and spectinomycin (100 μg/ml). Leaves of four to five-week-old N. benthamiana plants were co-infiltrated with an A. tumefaciens strain carrying the P19 silencing suppressor [28] in combination with strains harbouring bait or prey fusion constructs resuspended in infiltration buffer (10 mM MES, 10 mM MgCl2, and 150 μM acetosyringone, pH 5.6) to an OD of 0.3 per A. tumefaciens strain.

Confocal fluorescence microscopy
Microscopy was performed at 2-3 days after Agrobacterium-infiltration using a Zeiss LSM 700 laser scanning microscope. Leaf sections were incubated in propidium iodide (PI) solution (5 mg/ml) for 7-10 min prior to imaging to stain the cell wall. If no cytoplasmic strands were observed over multiple samples and the fluorescence signal was only observed in the same plane as the propidium iodide signal, the localization was considered plasma membrane. Excitation of Cyan Fluorescent Protein (CFP), Yellow Fluorescent Protein (YFP) and PI was done at 405 nm, 488 nm and 555 nm respectively. Emitted light of CFP and YFP was captured using a 490-555 nm band-pass filter, whereas emitted light of PI was captured using a 560 nm long pass filter.

Effector interaction screening identifies new and known target candidates
To investigate the molecular mechanisms underlying lettuce susceptibility to B. lactucae infection, we selected 46 previously identified candidate effectors from B. lactucae isolate Bl:24 [11,12,16] to be used as bait in Y2H screens ( Table 1). Orthologs of 34 effectors were found in isolate SF5 [13]. Consistent with the biotrophic lifestyle of B. lactucae, the vast majority of these candidate effectors are classified as RXLR-like effectors; when we started the Y2H screen only a single Crinkler (Bremia lactucae Crinkler; BLC) had been discovered. The RXLR-like effectors can be subdivided in two groups: 1) effectors with an RXLR motif (BLR effectors) or RXLR-like motif such as GXLR (BLG effectors) and QXLR (BLQ effectors), and 2) effectors that show homology to RXLR effectors but lack an RXLR-like motif, the BLN (Bremia lactucae No RXLR motif) effectors. All of the BLN effectors and the majority of the effectors with an RXLR(-like) motif contain an EER(-like) motif. The effectors ranged in size from 65 to 814 amino acids with an average length of 248 aa.
Despite a lack of general sequence conservation between all effector proteins within a species, it was found that about 44% of the P. infestans effectors, 26% of H. arabidopsidis effectors and 18% of Plasmopara halstedii RxLR effectors contain WY-domains that form a structurally conserved α-helical fold [23,29,30]. Analysis of the 46 B. lactucae effectors revealed predicted WY domains in 7 effectors (15%) ( Table 1). Among the potential WY domain-containing effectors are BLN06 and BLN08 that lack an RXLR motif, but are nevertheless recognized in specific lettuce lines [12,16], thereby demonstrating effector activity.
To identify lettuce proteins that are targeted by B. lactucae effectors a Y2H screen was performed. First, the coding sequences of the 46 B. lactucae effectors lacking the signal peptideencoding part were cloned in frame with the GAL4-DBD in the bait vector pDEST32. Of the 46 bait constructs three showed auto-activation in the Y2H system. The remaining 43 effectors were used as baits in Y2H screens with a prey library composed of L. sativa cv. Olof complementary DNA (cDNA). To promote representation of lettuce transcripts relevant to host-pathogen interactions, lettuce seedlings were challenged with compatible or incompatible B. lactucae isolates or treated with a SA-analog before RNA extraction. To eliminate false positives, identified interactors from lettuce were carefully scrutinized. Firstly, coding sequences that were out of frame with the GAL4 activation domain-encoding sequence were discarded.
Secondly, prey constructs that tested positive for autoactivation of the HIS3 reporter in the absence of the corresponding bait construct were also removed. Thirdly, bait and prey plasmids were retransformed in yeast to test if the interaction could be confirmed. These selection steps resulted, predominantly, in the elimination of candidate effector interactors that were identified only once. In contrast, most candidate effector interactors identified in two or more yeast colonies proved reliable interactors. Therefore, all interactors that were identified only once were omitted from further analysis. Ultimately, the library screens and subsequent validation steps resulted in interactors for 21 effectors (46%) ( Table 1) providing a set of 46 unique interacting lettuce proteins ( Table 2). Using domain prediction and orthologous relationships to Arabidopsis proteins, nine lettuce proteins were given gene names (S1 and S2 Files).
Interactions between effectors and lettuce proteins were further classified according to the number of unique colonies corresponding to the same lettuce gene and the interaction strength based on reporter gene activation (Fig 1). Due to the stringent selection criteria, only two weak (activation of HIS3 reporter only) interactions (3%) were identified, 25 interactions (41%) were classified as intermediate (activation of HIS3 reporter in the presence of 2 mM 3AT) and 34 interactions (56%) were strong (activation of both the HIS3 and ADE2 reporters). The vast majority of interactions between effectors and lettuce interactors were highly specific: only eight preys (17%) interacted with multiple effectors. LsRTNLB05, a reticulon-like protein, stood out from other lettuce interactors due to its interaction with five effectors and a combined total of 104 yeast colonies ( Table 2). The most yeast colonies, 50, within a single effector screen were identified with effector BLR18 for LsCSN5 that encodes COP9 signalosome subunit 5 (CSN5). However, LsCSN5 also displayed weak activation of the HIS3 reporter in the presence of empty bait vector.
The most represented family of proteins in the screen is the prenylated rab acceptor (PRA1) family for which fragments of six members were identified and three members passed all selection criteria. PRA1 genes encode small transmembrane proteins that localize to the secretory pathway in Arabidopsis and are proposed to play a role in vesicular trafficking in plants [31]. Also, they have previously been described as interactors of G. orontii effector candidates in Arabidopsis [9]. In lettuce the PRA1 protein family is composed of 17 members. The six Y2H identified PRA1 proteins were found in four clades of the phylogenetic tree (Fig 2A). To determine the specificity of the interactions between B. lactucae effectors and PRA1 proteins, a targeted Y2H assay was performed. All six identified prey constructs containing lettuce PRA1 gene fragments were cotransformed with three interacting and one non-interacting B. lactucae effector in yeast and plated on selective medium of increasing stringency. The negative control BLR09 did not interact with any of the PRA1 members. The interactions between effectors BLR27, BLR32 and BLN01 with individual PRA1 proteins as identified in the library screens were confirmed. Furthermore, BLR27 and BLR32 interacted with more PRA1 proteins than determined by the library screen, indicating that the Y2H screening was not exhaustive and not all possible interactions were captured. In contrast, BLN01 interacted robustly with LsPRA1.B1 but only weakly with other PRA1 proteins (Fig 2B). Two of the three PRA1 proteins that passed all selection criteria belong to clade F. Interestingly, the Arabidopsis PRA1 proteins that interacted with G. orontii and H. arabidopsidis effectors also belong to clade F [9].  Twelve plant interactors were selected for further exploration in planta, including the top ten ranked effector interactors by total number of yeast colonies. In addition, LsBPM3 and LsERF093 were chosen because of their predicted functions in targeting proteins for degradation and transcriptional regulation respectively, as will be described in more detail later.
To further explore the selected effector interactors, a closer look was taken at the prey plasmid-encoded gene fragments. DNA sequences obtained by Sanger sequencing using a vector specific forward primer were aligned to the corresponding lettuce coding sequences. A vector specific reverse primer could not be used due to the presence of poly(A)tails in prey inserts leading to slippage of the polymerase during sequencing. Therefore, information on the 3' end of inserts >800bp was not obtained. Lettuce gene fragments that included the start codon of the lettuce coding sequence (Fig 3) were detected for eight preys. Yeast clones of the remaining four preys only contained fragments that started downstream of the predicted start codon. The use of a cDNA library that contains both full-length coding sequences and gene fragments likely increased the number of identified protein-protein interactions.

Colocalization of effectors and their plant interactors
As proteins that are localized in the same subcellular compartment in planta are more likely to be true interactors, we set out to determine the subcellular localization of 15 B. lactucae effectors and 12 plant interactors using confocal fluorescence microscopy. The full-length coding sequences of the selected lettuce genes were cloned and fused downstream of CFP. Effector sequences were fused to YFP. The fusion proteins were produced in leaves of Nicotiana benthamiana using Agrobacterium-mediated transient expression. Immunodetection, using GFP-antibodies showed that for most proteins a fusion product of the expected size was produced by overexpression in planta (Figs 4 and 5). Detection of YFP-BLR18 showed a large fraction of free YFP and a minor fraction corresponding to the intact fusion protein, suggesting that the observed nucleocytoplasmic signal may result from free YFP. For other effector fusions that do not display an even nucleocytoplasmic localization pattern, it is likely that the observed free YFP on the immunoblots originates from degradation occurring during protein extraction. Of the 15 effectors described here, four localized exclusively to the cytoplasm (BLN01, BLR11, BLR20, and BLR27) and two (BLG02 and BLR18) showed both cytoplasmic and nuclear localization (Fig 4 and Table 3). In contrast, YFP-BLR28 and BLR38-YFP localized solely to the nucleus (also see [12]), as expected because they are predicted to have nuclear localization signals (NLSs) (S2 Table). A predicted C-terminal transmembrane domain is present in six effectors (Table 1 and S3 Table) and these were thus expected to localize to membranes. We were unable to detect BLN03 and BLN04 C-or N-terminal fusion proteins, resulting in localizations for 13 of the 15 tested effectors (Fig 4). YFP-BLR05 and YFP-BLR09  Arabidopsis PRA1 domain-containing proteins. Sequences were aligned using Clustal Omega. Tree construction was performed in MEGA 7.0 using Neighbor-Joining with partial deletion of sites with less than 95% coverage. Nodes with less than 50% confidence score based on 1000 bootstrap replicates were removed. The previously reported interactions between Hyaloperonospora arabidopsidis and Golovinomyces orontii effectors HaRxL74 and OEC65 and Arabidopsis PRA1 proteins have been added. An asterisk ( � ) behind the protein indicates it was included in the targeted Y2H. B) A targeted Y2H was predominantly labeled the endoplasmic reticulum (S1 Fig), whereas YFP-BLR08 was associated with ring-like structures of various sizes in line with our previously reported findings [32]. YFP-BLR12 appeared as punctate structures. Upon prolonged overexpression, YFP-BLR12 also labeled the endoplasmic reticulum suggesting that the punctate structures represent components of the secretory pathway, that, however, requires confirmation using appropriate markers. Interestingly, BLG03 that is recognized in Dm2 lettuce lines [11], does not contain predicted transmembrane domains, but YFP-BLG03 was associated with the plasma membrane (S1 Fig) similarly to BLN06-YFP and BLR40-YFP [12]. It is possible that post-translational modifications (S4 Table) contribute to observed membrane localizations.
Subsequently, the localization of tagged lettuce proteins was analyzed. Seven of the twelve tested proteins localized to the cytoplasm and/or nucleus (Fig 5 and Table 3). LsFLX-like2 and Lsa021294.1 localization was restricted to small, punctate structures in the cytoplasm. In line with the absence of signal peptide or transmembrane domain in LsFLX-like2 and Lsa021294.1, no ER localization was observed. The structures are thus more likely to present cytoplasmic protein complexes than components of the secretory pathway. However, for now, their origin remains undetermined. We were unable to detect C-or N-terminal LsRTNLB05 fusion proteins, resulting in localizations for 11 of the 12 interactors from lettuce (Fig 5). LsNAC069 and Lsa008464.1 labelled the endoplasmic reticulum (S2 Fig).
Upon co-expression of the effectors with lettuce proteins, complete or partial colocalization was observed for 10 out of 13 combinations, entailing 11 effectors and 10 lettuce interactors performed with yeast isolated prey plasmids containing gene fragments of six PRA1 family members using B. lactucae effectors BLR27, BLR32 and BLN01 as bait. BLR09 was included as a negative control bait. Dashed line indicates the PRA1 protein did not pass all Y2H selection criteria, a continuous line indicates the PRA1 proteins passed the Y2H selection criteria. Left: permissive plate containing histidine. Middle: moderately selective plate lacking histidine. Right: strongly selective plate lacking adenine. The positions of previously identified interactions in the library screens are indicated with grey blocks for selection on SC medium lacking histidine.
https://doi.org/10.1371/journal.pone.0226540.g002   4. Subcellular localization and stability of 13 B. lactucae effectors in N. benthamiana. Effectors were fused N-terminally to YFP at the predicted signal peptide cleavage site, with the exception of BLR38 that was cloned as a C-terminal fusion protein, and transiently expressed in N. benthamiana using Agrobacterium. a) Images were taken 2-3 dpi. Bars = 10 μm. n = nucleus b) Immunoblots of the fusion proteins indicating their size and stability. Most effector fusion proteins are predominantly present in monomeric form with only a single band at the expected height of the fusion protein and occasionally lower bands that may represent cleavage products. YFP-BLR20 forms an exception as besides a band corresponding to the monomer, equally intense bands are present at twice and four times the weight of YFP-BLR20 indicating that it forms (stable) dimers and multimers under the denaturing conditions used. Grey arrows indicate the monomeric size of the fusion proteins, black arrows indicate multimers and open arrows indicate free YFP. Data on BLR05 and BLR09 have been published earlier [32]. https://doi.org/10.1371/journal.pone.0226540.g004
To appreciate the relocalization phenomenon we present the localization of singly expressed proteins besides the co-expressed proteins for a number of interactions (Fig 7). Relocalization to the nucleus of effector BLG02 (mainly cytoplasmic, although in some cells also weakly nuclear) occurred in the presence of the nuclear-localized protein CFP-LsBPM3 (Fig  7). The cytoplasmic effector YFP-BLR20 relocalized to the nucleus (Fig 7) upon co-expression of nuclear localized CFP-LsERF093 and the intensity of the YFP nuclear signal was dependent on CFP-LsERF093 signal intensity.
In other cases, effector expression led to relocalization of the interactor. Effector BLR38-YFP induced a relocalization of its interactor CFP-LsFLX-like2 from punctate cytoplasmic structures to the nucleus (Fig 7). Also, nuclear-localized effector YFP-BLR28 induced relocalization of CFP-LsDjA2 to the nucleus (Fig 7). LsDja2 was predominantly cytoplasmic and in some cells nucleocytoplasmic upon single expression, but co-expression with BLR28 induced a shift towards nucleocytoplasmic localization. In summary, ten of the thirteen effector-lettuce protein pairs colocalized in N. benthamiana. Furthermore, relocalization occurred upon expression of four effector-lettuce protein pairs providing evidence that the Y2H-identified interactions also occur in planta.

Features of the B. lactucae effectors
RxLR effectors are restricted to oomycetes belonging to the clade of the Peronosporales. In each species a number of these effectors contain structurally conserved WY domains that Table 3. Subcellular localization of FP-fusions of 11 effectors and 10 corresponding lettuce proteins, expressed individually or together to determine possible co-or re-localization.  adopt an α-helical fold supported by W/Y/L sequence motifs [23,30]. The WY-domain is present in 44% of the Phytophthora RxLR effectors, but is less abundant in downy mildews: 18% of the Plasmopara halstedii [29] and 26% of the H. arabidopsidis [23] RxLR effectors contain WY domains. Our analysis revealed that from the set of 46 B. lactucae effectors originating from isolate Bl:24 seven (15%) effectors have predicted WY domains. These seven effectors were also identified in isolate SF5 [33]. The differences in WY-domain abundance may reflect a bias in the WY-domain algorithm towards Phytophthora sequences; other downy mildew effectors may contain a, so far, unrecognized conserved fold.

Yeast two-hybrid interactions between B. lactucae effectors and lettuce proteins
The cDNA library-based Y2H screen resulted in lettuce interactors for 21 (46% of screened) effectors. These effectors interacted on average with three lettuce proteins. The vast majority (83%) of identified lettuce proteins interacted only with a single B. lactucae effector. Previously published screenings with C. elegans and human fragment libraries resulted in interactors for 37% and 31% of bait proteins respectively corresponding to an average of 2.2 and 2 interactors per bait [34,35]. An average of 3.4 interactors per effector was obtained in screens with effectors from G. orontii, H. arabidopsidis and P. syringae against a library of~8000 immune- related full-length Arabidopsis proteins [8,9]. The number of obtained interactors in our screen is thus comparable to other published Y2H screens. Based on the identification of Arabidopsis proteins that interacted with effectors from three pathogens [9], it has been proposed that effectors converge on conserved host proteins. This proposition fits with insights into the mechanisms by which independent bacterial type III effectors converge on immune-related proteins such as MAPK proteins [36], SERK3/BAK1 [37][38][39][40][41] and RIN4 [42]. We were interested to determine if those proteins identified as major hubs, i.e. interacting with effectors from multiple pathogens, would also emerge in our screens with B. lactucae effectors.
The list of Arabidopsis hubs was dominated by TCP transcription factors [9]. Specifically, TCP13, TCP14 and TCP15 interacted with effectors originating from all three pathogens, and TCP19 and TCP21 interacted with effectors from at least one pathogen [9]. TCPs operate as transcriptional activators or repressors in plant growth and development [43,44]. Lettuce TCP family members were also detected as interactors of B. lactucae effectors. However, the effector-TCP interactions weakly activated the HIS3 reporter, were frequently observed as single colonies in Y2H screens and TCPs showed autoactivation in the Y2H system. Weßling and colleagues also classified Arabidopsis CSN5A as a hub due to its interaction with 12 P. syringae effectors, 11 H. arabidopsidis effectors and 9 G. orontii effectors in Y2H screens [8,9]. In our library screens, lettuce CSN5 was identified with two B. lactucae effectors, but also showed weak activation of the HIS3 reporter in the presence of an empty bait vector. Interaction between CSN5 and the GAL4 DNA binding domain was previously reported using multiple GAL4-DBD based vectors and yeast strains [45][46][47]. Rejecting CSN5 as false positive because of stronger reporter gene activation in the presence of B. lactucae effectors than empty vector is only partially defendable. Effectors may have unforeseen additive effects on reporter gene activation but these effects could be interaction independent. Thus, though some of the previously reported hubs may represent host proteins that play a prominent role in disease susceptibility, others may be false positives.

Several B. lactucae effectors localize to membranes in planta
We examined the localization of a subset of 15 B. lactucae effectors and assessed their stability using immunoblot. Analysis of the YFP-BLR20 fusion protein on an SDS-PAGE gel under denaturing conditions revealed multiple bands corresponding to monomeric, dimeric and multimeric forms of this effector. It is possible that a multimer represents the biologically active form of BLR20 as multimerization of two P. infestans effectors, PexRD2 and PiSF3, was shown to be required for interaction with their targets, a host MAP kinase and U-Box-kinase protein respectively [23,48,49]. The multimerization of BLR20 is possibly driven by the presence of predicted WY-domains that have been proposed to support oligomerization [23]. This was previously shown for PiSF3 by mutation of two residues facing each other across the αhelices of PiSF3 that disrupted the formation of oligomers [48]. Interestingly, multimerization of PexRD2 and PiSF3 was not demonstrated under denaturing conditions. Some transmembrane domains or transmembrane domain containing proteins give two bands corresponding to the monomer and multimer under denaturing conditions [50]. More research is required to determine if non-covalent interactions between hydrophobic interfaces of WY domains cause the observed multimerization or if other factors play a role.
B. lactucae effectors BLG03, BLN06 and BLR40, which are recognized and trigger HR in specific lettuce lines, localized to the plasma membrane [11,12]. Plant membranes were also targeted by 26% of 49 tested H. arabidopsidis effectors [51] and 12% of 76 tested Plasmopara viticola effectors [52]. Coiled-coil domains [53] and post-translational modifications such as N-myristoylation, could contribute to anchoring of effectors, lacking transmembrane domains, to the plasma membrane [51, 54,55]. Six effectors-BLN03, BLN04, BLR05, BLR08, BLR09 and BLR12 -were predicted to contain a single C-terminal transmembrane domain based on TMHMM analysis. Effectors BLR05, BLR08, BLR09 and BLR12 were indeed found to be associated with the endomembrane system: YFP-BLR05, YFP-BLR09 [32] and YFP-BLR12 strongly labelled the endoplasmic reticulum, whereas BLR08 was associated with ring-like structures of varying sizes. Recently, it has been shown that BLR05 and BLR09 colocalized with their interactor LsNAC069 at the endoplasmic reticulum [32]. The B. lactucae effectors were cloned without their signal peptide, which is required for co-translational ER targeting of transmembrane proteins by the signal recognition particle [56]. Instead membrane integration of tail-anchored proteins occurs post-translationally and is dependent on a C-terminal transmembrane domain that functions as a targeting signal. Some proteins with a moderately hydrophobic TM domain can insert unassisted in the ER, others with a highly hydrophobic TM domain require assistance. In yeast, the TM domain of tail-anchored proteins is recognized by components of the GET system including Get3 (TRC40 in mammals), delivered to the ER-resident Get1/Get2 receptor complex and inserted into the membrane [57][58][59][60]. Clearly, the ER localization of YFP-BLR05, YFP-BLR09 and YFP-BLR12 proves that these effectors associate with the membrane post-translationally. This immediately prompts the question how membrane-associated effectors are delivered to host cells. A role for extracellular vesicles in the delivery of soluble effectors has been hypothesized [61] and vesicular transport would provide a suitable vector for membrane-associated effectors as well. The Cterminal transmembrane domain of YFP-BLN03 and YFP-BLN04 may not have been sufficient for posttranslational insertion providing a possible explanation for the lack of fluorescence of these fusion proteins.
Effector BLG03 targeted the plasma membrane despite lacking a putative transmembrane domain. Post-translational modifications resulting in protein lipidation such as prenylation, N-myristoylation or palmitoylation can affect membrane-protein interactions [62]. Putative N-myristoylation sites were implicated in the membrane targeting of multiple bacterial type III effectors [55,63]. Alternatively, phospholipid conjugation [62] or phospholipid binding may contribute to membrane association. Multiple studies have explored the role of the N-terminal RXLR motif and C-terminal residues in phospholipid binding [64]. Though the relevance of phospholipid binding for effector uptake remains unclear, perhaps a role in effector localization in planta could be considered.

Co-expression of effectors and interactors induces protein relocalization to the nucleus
Studies on H. arabidopsidis and P. viticola RXLR effector localization revealed a preference for nuclear (including nucleocytoplasmic) localization (66% and 83% respectively) [51,52]. In our research, only two effectors-BLR28 and BLR38 -were strictly nuclear localized in N. benthamiana. Strikingly, B. lactucae RXLR effector BLR38 that is recognized in L. serriola LS102, induced a relocalization of its interactor LsFLX-like2 to the nucleus. LsFLX-like2 shows homology to the Arabidopsis FLOWERING LOCUS C EXPRESSOR (FLX) protein family of which two members are involved in flowering time control in Arabidopsis [65]. The punctate structures of CFP-LsFLX-like2 resemble the structures formed by cytoplasmic FLX homodimers in Arabidopsis [66]. Heterodimers of FLX with FRIGIDA or FRIGIDA ESSENTIAL 1 were restricted to the nucleus [66], suggesting that the observed relocalization of CFP-LsFLX-like2 upon co-expression with BLR38-YFP may reflect a different interaction state. The identification of LsFLX-like2 in a screen for B. lactucae effectors may hint towards a role of FLX-like family members in the integration of environmental signals in flowering time regulation. Bacterial, fungal and downy mildew infection induce early flowering of Arabidopsis plants [67,68] although the molecular mechanism by which biotic stress is integrated in flowering time regulation remains poorly understood. Thus, it would be interesting to explore if B. lactucae infection affects regulation of flowering time in lettuce. BLR28 also induced relocalization of its interactor, DnaJ protein LsDjA2, to the nucleus. DnaJ proteins function as co-chaperones to 70 kDa heat shock proteins to aid in protein folding, relocalization and degradation [69].
Furthermore, two predominantly cytoplasmic effectors, BLG02 and BLR20, displayed nucleocytoplasmic localization when co-expressed with their interactors, LsBPM3 and LsERF093. LsERF093 contains an AP2/ERF domain that is typically found in members of the AP2/ERF transcription factor family. These proteins are known to be involved in responses to biotic and abiotic stress in Arabidopsis, tomato and rice [70,71]. LsBPM3 contains a BTB/POZ (broad complex, tram track, bric-a-brac ⁄POX virus and zinc finger) domain that is also found in 80 Arabidopsis proteins and is known to mediate protein-protein interactions. Specifically, the BTB/POZ domain acts as a substrate-specific adaptor for Cullin-RING E3 ubiquitin Ligases (CRLs) that ubiquitinate proteins targeted for degradation by the proteasome. Recruitment of substrates may occur via a Meprin and TRAF homology (MATH) domain [72,73]. The Arabidopsis MATH-BTB/POZ protein BPM3 localizes to the nucleus and promotes degradation of transcription factor ATHB6 that negatively regulates abscisic acid signaling [74]. Furthermore, BPMs interact with APETALA 2/ethylene-responsive element binding factor (AP2/ERF) transcription factors [75].
The relocalization events that we observed, have two important implications. First, they suggest that physical interactions between RXLR effectors and interactors occur in planta, which may affect the activity of the interactor and consequently, disease development. This principle was recently demonstrated for P. sojae effector Avh52 that recruits a cytoplasmic host transacetylase, GmTAP1, into the nucleus. Nuclear-localized GmTAP1 is able to transacetylase histones, which enhances susceptibility of N. benthamiana to P. capsici [76]. Also, Arabidopsis protein RD19 that was discovered as interactor of Ralstonia solanacearum type III effector Pop2 in a Y2H screen, relocalized to the nucleus in the presence of the effector and physically associated with Pop2. The effector target, RD19, was required for the resistance protein RRS1-R mediated immune responses [77] Thus, relocalization events are often associated with physical interactions between effector and interactor, and effector-target interactions can affect disease susceptibility. Functional analysis of the lettuce interactors is required to provide further insights into the mechanisms underlying B. lactucae susceptibility.
The second implication is that screening by Y2H is a useful method to identify potential host targets. However, the biological relevance of the observed interactions needs to be demonstrated in follow-up experiments and there is no universal rule (yet) for selecting the most promising candidates. We investigated LsFLX-like2/ BLR38 and LsDjA2/ BLR28 because they belonged to the top ten ranked interactions in the Y2H screen. In contrast, BLG02/ LsBPM3 and BLR20 / LsERF093 were selected based on literature demonstrating a role for the target candidate protein families in immune responses. It should furthermore be stressed that neither of these four interactors function as hub in our Y2H screen: all of them interacted with a sole effector. Conversely, we have previously demonstrated that small networks identified in Y2H screens can be valuable i.e. LsNAC069 interacted with four-BLN04, BLR05, BLR08 and BLR09-effectors in Y2H assays and was demonstrated to affect plant responses to both biotic and abiotic stress [32]. To conclude, our Y2H screens have uncovered multiple potential effector targets whose precise function we are just beginning to unravel. Supporting information S1