# A Salivary Effector of the Pea Aphid Interacts with Pea Proteins and Enhances Its Performance on the Host Plant

Po-Yuan Shih, Stéphanie Le Bras, Rémi Ollivier, Hélène Boulain, Stéphanie Morliere, Yannick Outreman, Jean-Christophe Simon, Akiko Sugio

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Po-Yuan Shih, Stéphanie Le Bras, Rémi Ollivier, Hélène Boulain, Stéphanie Morliere, et al.. A Salivary Effector of the Pea Aphid Interacts with Pea Proteins and Enhances Its Performance on the Host Plant. Molecular Plant-Microbe Interactions, 2025, 10.1094/mpmi-08-24-0089-r hal-05166019v2

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RESEARCH

# A Salivary Effector of the Pea Aphid Interacts with Pea Proteins and Enhances Its Performance on the Host Plant

Po-Yuan Shih,<fnref n="1" /> Stéphanie Le Bras,<fnref n="1" /> Rémi Ollivier,<fnref n="1" /> Hélène Boulain,<fnref n="1" /> Stéphanie Morlière,<fnref n="1" /> Yannick Outreman,<fnref n="2" /> Jean-Christophe Simon,<fnref n="1" /> and Akiko Sugio<fnref n="1" />,†

Accepted for publication 14 May 2025.

Aphids have intricate interactions with their host plants, but the molecular mechanisms behind these interactions remain largely unknown. The pea aphid, *Acyrtosiphon pisum*, is a legume specialist that forms a complex of several biotypes, each specialized in feeding on a few legume species. Aphids inject a cocktail of salivary effector proteins into plants to suppress plant immunity and promote their performance, such as fecundity. Previous studies showed that subsets of salivary effector genes of *A. pisum* are differentially expressed between the pea- and alfalfa-adapted biotypes. We hypothesized that the salivary effector genes that are important for *A. pisum* to feed on pea (*Pisum sativum*) are highly expressed in the pea-adapted biotype compared with non-adapted ones and selected such genes for functional characterization. We examined 10 candidate genes and found that expression of the salivary gene *LOC100159932* (*Ap4*) in pea leaves increased the fecundity of the *A. pisum* pea biotype. A yeast two-hybrid screening using *Ap4* as a bait identified two pea proteins named PsBPL1 and PsBPL2 that showed high homology with Arabidopsis BPL (Binding Partner of ACD11-Like) proteins, which are conserved in plants and fungi and known to be involved in plant immunity. GFP-tagged Ap4 proteins produced puncta in *Nicotiana benthamiana* cytoplasm, and a bimolecular fluorescence complementation experiment confirmed the interaction of Ap4 and PsBPL1 and PSBPL2 in planta. These results highlight Ap4's role as an effector and

suggest the involvement of BPL proteins in pea-aphid interactions.

*Keywords:* Binding Partner of ACD11, effector, host adaptation, plant immunity, protein-protein interaction

Insect herbivores utilize host plants as food and habitats, and most of them are specialized to feed on a limited number of plant species (Forister et al. 2015). However, the mechanisms that are involved in the interactions of insects and their host plants are little known. Aphids are one of the serious pests of many crops, as they take up phloem sap and transmit plant-pathogenic viruses. During the feeding process, aphids secrete saliva that contains effectors (virulence factors) that alter the plant-aphid interactions (Elzinga and Jander 2013; Rodriguez and Bos 2013; Shih et al. 2023). Several effector proteins have been shown to enhance aphid fitness by reducing plant immunity. For example, a *Myzus persicae* effector, Mp55, reduces aphid-induced plant defense responses, such as callose deposition and production of hydrogen peroxide, and promotes aphid fecundity in Arabidopsis (*Arabidopsis thaliana*) and *Nicotiana benthamiana* (Elzinga et al. 2014). A *Macrosiphum euphorbiae* effector, Me47, can metabolize toxic isothiocyanates of tomato and enhances aphid fecundity (Kettles and Kaloshian 2016). An *Aphis craccivora* effector, AcDCXR, is thought to reduce toxic methylglyoxal and enhances reproduction of this aphid on pea (MacWilliams et al. 2020). Interestingly, some of these characterized salivary effectors appear to function in a species-specific manner, suggesting their roles in aphid host specificity (Pitino and Hogenhout 2013).

Recently, specific plant protein targets of aphid salivary effectors have been identified as part of an effort to understand their role in the interaction with host plants. *M. persicae* effector Mp1 was shown to interact with Vacuolar Protein Sorting associated protein52 (VPS52) of its host plants, Arabidopsis (AtVPS52) and potato (*Solanum tuberosum*, StVPS52), in a yeast two-hybrid (Y2H) system but did not interact with the orthologous proteins of the plants on which it does not perform well (*Medicago truncatula* and *Hordeum vulgare*) (Rodriguez et al. 2017). Ectopic expression of StVPS52 in *N. benthamiana* reduces the fecundity of *M. persicae* on the plant, and *M. persicae* infestation reduces the AtVPS52 protein level in Arabidopsis. These results suggest that VPS52 positively contributes to the plant defense against the aphid. Mp1 was also identified as an interactor of Arabidopsis phloem protein AtPP2-A1 by a pull-down assay, and the interaction of the two proteins in *N. benthamiana* and Arabidopsis cytoplasm was confirmed by bimolecular fluorescence complementation (BiFC) analysis

Current address for P.-Y. Shih: Department of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom.

Current address for R. Ollivier: Center for Quantitative Genetics and Genomics, Aarhus University, Slagelse, Denmark.

Current address for H. Boulain: Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland.

**Funding:** This work was funded by Agence Nationale de la Recherche (ANR): Plant2Pro-2018-CharaP, Plant2Pro-2022-R2V2, and ANR-P-Aphid (ANR-18-CE20-0021-01) to A. Sugio and ANR-Mecadapt (ANR-18-CE02-0012) to J.-C. Simon. Ph.D. grant for R. Ollivier was provided by Institut national de recherche pour l'agriculture, l'alimentation et l'environnement (INRAE) and Région Bretagne.

**e-Xtra:** Supplementary material is available online.

The author(s) declare no conflict of interest.

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<footnote n="1">¹IGEPP, INRAE, Institut Agro, Université de Rennes, 35653, Le Rheu, France</footnote>

<footnote n="2">²IGEPP, INRAE, Institut Agro, Université de Rennes, 35000, Rennes, France</footnote>

<footnote>†Corresponding author: A. Sugio; akiko.sugio@inrae.fr</footnote>
(Wang et al. 2021). The phloem feeding time of *M. persicae* was reduced on the Arabidopsis overexpressing AtPP2-A1 and increased on the *atpp2-a1* mutant, supporting the involvement of AtPP2-A1 in Arabidopsis defense against the aphid (Wang et al. 2021). Finally, Y2H screening showed that an *M. euphorbiae* effector, Me10, interacts with the tomato 14-3-3 isoform 7 (TFT7). *TFT7* expression is induced upon *M. euphorbiae* infestation and confers non-host resistance to *Aphis gossypii* in tomato (Chaudhary et al. 2019). In these examples, the mechanisms by which the aphid effectors interfere with plant defenses and enhance aphid performance are still unknown, but the identification of aphid effector targets has opened new directions for studying plant immune systems against the aphids.

The pea aphid, *Acyrthosiphon pisum*, is specialized to feed on legume species and considered a serious pest of various crops. This aphid forms a species complex of at least 15 biotypes, each of which is genetically distinct and specialized to feed on a limited number of legume species (Peccoud et al. 2009, 2015). Previous genome analysis of different *A. pisum* biotypes found that salivary effector candidate genes are enriched in the regions that are highly differentiated between these biotypes (Nouhaud et al. 2018). Transcriptome studies further revealed that some salivary effector candidates are differentially expressed in *A. pisum* biotypes (Boulain et al. 2018; Eyres et al. 2016). In addition, co-infection of the *A. pisum* pea-adapted biotype (hereafter, pea biotype) can increase the phloem feeding time of the alfalfa-adapted biotype (hereafter, alfalfa biotype) and consequentially increases the survival rate and fecundity of the alfalfa biotype on the pea plant (*Pisum sativum*), which is a non-host plant for the alfalfa biotype (Shih et al. 2023). These results strongly support the hypothesis that distinct *A. pisum* biotypes utilize specific salivary effectors to enhance their performance on specific host plants. In this study, we explored this hypothesis on the pea and alfalfa biotypes of *A. pisum*, for which we generated various genetic and expression data (Boulain et al. 2019; Eyres et al. 2016; Nouhaud et al. 2018) and developed tools for functional characterization (Guy et al. 2016). We hypothesized that *A. pisum* salivary genes that are highly expressed in the pea biotype compared with the alfalfa biotype may be particularly important for the pea biotype to feed on pea plants. We also hypothesized that those secreted salivary proteins may interact with plant proteins to exert their functions. Our previous analyses identified a set of candidate salivary genes in *A. pisum* that were expressed in the salivary glands, predicted to encode secreted proteins and up-regulated in the pea biotype compared with the alfalfa biotype (Boulain et al. 2018, 2019). In this study, we selected the candidate salivary genes that are highly upregulated in the pea biotype and examined their functions in pea and *A. pisum* interactions. We showed that one of the tested salivary effectors improved the performance of the pea biotype of *A. pisum* and identified its protein targets in the pea plant.

# Results

## Selection of salivary effector candidates for the functional characterization

Earlier expression data of salivary effector candidates in the *A. pisum* pea and alfalfa biotypes, each represented by three genetically distinct aphid clones (Boulain et al. 2019), were used to select candidate salivary effector genes for functional characterization. Our previous study identified 3,546 genes that are expressed in the salivary glands of the *A. pisum* pea biotype clone P123 or the alfalfa biotype clone LSR1 and encode the proteins that are predicted to be secreted (Boulain et al. 2019). A transcription study of head samples (which contained salivary glands) of the pea and alfalfa biotypes, each represented by three aphid clones (clones ArPo58, P123, S1PS02 for the pea

biotype and clones LSR1, LL01, L84 for the alfalfa biotype), showed that 103 out of 3,546 genes were upregulated in the pea biotype compared with the alfalfa biotype (Boulain et al. 2019). As some of the 103 genes may have a ubiquitous expression throughout aphid body and may not have specific functions in plant-aphid interactions, we reduced the list to 67 genes that are specifically upregulated in the salivary glands compared with the digestive tract, using the data presented in Boulain et al. (2019) (Supplementary Table S1). In this earlier work, the digestive tract was selected to examine organ-specific expression of the genes due to the ease of dissecting this organ. From the 67 genes, we retained 23 genes that were highly and differentially expressed between the salivary glands of P123 and LSR1 clones according to the following criteria: (i) their expression exceeded 50 counts per million (CPM) in the pea biotype clone P123, (ii) their expression was at least twofold higher in P123 than in LSR1, (iii) the false discovery rate (FDR) threshold for detecting differences in gene expression level between P123 and LSR1 salivary glands was lower than 0.05, and (iv) variability in expression between the three clones of each biotype was less than 30% of their average expression value, which resulted in 17 candidate effector genes. One gene, *LOC100571683*, was removed from the updated genome annotation by NCBI. Of the remaining 16 candidates, two (*LOC100571965* and *LOC100572516*) were predicted to be secreted without having a classical secretion signal according to the prediction by SecretomeP (Bendtsen et al. 2004) (Supplementary Table S1). Therefore, we focused on the remaining 14 candidate effectors with a classical secretion signal peptide. The gene *C002* (*LOC100167863*), which was among our shortlist of 14 genes and did not affect the fecundity of the pea biotype when expressed in pea leaves in a prior work (Guy et al. 2016), was not assessed in this study. We were unable to clone the complementary DNA (cDNA) of *LOC100573120*, *LOC100161530*, and *LOC100163506*, although they met all the criteria we used. Eventually, 10 candidate salivary effector genes were cloned and analyzed. Of these 10 candidates, three genes (*LOC100159932*, *LOC100302326*, and *LOC1000160421*) showed homology with previously studied aphid salivary genes (*M. persicae Mp16* and *Mp31* and *Sitobion miscanthi Sm9723*, respectively) (Bos et al. 2010; Zhang et al. 2022).

## The effector Ap4 promotes *A. pisum* pea biotype fecundity on pea

To examine the effect of the candidate salivary effector proteins secreted into pea cells, the cDNA of the candidate genes without the 5' sequence encoding the N-terminal secretion signal was individually expressed in pea leaves using *Agrobacterium*-mediated transient expression system, and the effect of the gene expression on aphid performance was examined (Guy et al. 2016). The pea biotype clone ArPo58 was used for this assay, as it was used in the previous characterization of the salivary gene *Ap25* (Guy et al. 2016) and the transcriptomic analysis (Boulain et al. 2019). The survival rate of the pea biotype did not show a significant difference on leaves expressing any of the salivary gene candidates compared with control leaves expressing enhanced green fluorescent protein (eGFP) (Fig. 1A; Supplementary Data). However, aphid fecundity was significantly increased on leaves expressing *LOC100159932* (*ACYPI001271*, named *Ap4* in Bos et al. 2010) (Fig. 1B; Supplementary Data). The survival rate of the alfalfa biotype clone, LL01, was not affected by the expression of any of the salivary gene candidates, including *Ap4*, compared with eGFP (Supplementary Fig. S1; Supplementary Data). LL01 did not survive long enough on pea plants to allow for an assessment of the effect of *Ap4* expression on its fecundity.
## Ap4 is an aphid-specific gene

Ap4 (LOC100159932) was first identified by analyzing the expressed sequence tag (EST) data of the salivary glands of the A. pisum alfalfa biotype LSR1 clone, and the encoded protein was also detected by proteomics analysis of the salivary glands of alfalfa biotype clones LL01 and LSR1 (Carolan et al. 2011; Boulain et al. 2018). Ap4 is 645 bp, encoding a protein of 214 amino acids, and is on average four times more expressed in heads (that contain salivary glands) of the pea biotype compared with the alfalfa biotype (Boulain et al. 2019) (Supplementary Table S1). No non-synonymous nucleotide difference between the pea and alfalfa biotypes was detected based on the genome sequence data in Nouhaud et al. (2018), and the Ap4 proteins encoded by clones LL01 and P123 were the same. By BLASTing the Ap4 protein sequence against the predicted protein data from aphid genome sequences, presumptive orthologs of Ap4 were identified in 10 aphid species in addition to A. pisum: M. euphorbiae, M. persicae, Rhopalosiphum padi, Rhopalosiphum maidis, Melanaphis sacchari, Myzus cerasi, Pentalonia nigronervosa, Aphis glycines, A. craccivora, and A. gossypii (Fig. 2). All these species belong to the Aphidinae subfamily of the family Aphididae. There was no hit with the predicted proteins of *Daktulosphaira vitifoliae*, which belongs to the Phylloxeridae family. One protein sequence of *Eriosoma lanigerum*, which belongs to the Eriosomatinae subfamily of Aphididae, showed low identity (22%, similarity 47%) with Ap4, and the e-value (0.017) was higher than our cutoff value. These results supported the previous observation that Ap4 is specific to the Aphididae (Boulain et al. 2018) and, in particular, the Aphidinae subfamily. The 10

Ap4 presumptive orthologous proteins showed 35 to 77% identity with Ap4, and a conserved signal peptide was predicted in all the proteins except the proteins of *M. cerasi*, *A. craccivora*, and *A. gossypii* by SignalP 5.0 (Almagro Armenteros et al. 2019). Orthologs of Ap4 in *A. craccivora* and *A. gossypii* were predicted to encode shorter proteins than the other orthologs. These two orthologs are located at the end of the scaffold of their respective aphid genome and are likely poorly annotated. DeepTMHMM (Hallgren et al. 2022) and PredGPI (Pierleoni et al. 2008) predicted that none of these proteins was membrane-bound, and WoLF PSORT (Horton et al. 2007) and DeepLoc 2.0 (Thumuluri et al. 2022) predicted that these proteins, except the ones from *A. craccivora* and *A. gossypii*, were secreted to the extracellular space (Supplementary Table S2).

## Ap4 interacts with pea PsBPL proteins in yeasts

The Ap4 protein may be secreted by *A. pisum* into plant cells and physically interact with plant proteins to exert its function and increase aphid performance. To understand how Ap4 functions in plants, we aimed to identify pea proteins that physically interact with Ap4. A pea cDNA library generated from the mixture of aphid-infested and noninfested pea plants was used for a Y2H screening using Ap4 as a bait. Fragments encoding a total of 16 pea proteins were identified as candidate Ap4 interactors, and eight of them were selected for further study by considering the predicted biological scores (Formstecher et al. 2005), the predicted function, and the length of the coding sequence (<1.5 kb) (Supplementary Table S3). The full-length cDNAs of the eight pea genes were cloned from the pea cDNA used for

Fig. 1. Effect of the expression of the candidate salivary effector genes on the A, survival and B, fecundity of the pea biotype (ArPo58) on pea. Data are shown in box plots and analyzed by a generalized linear mixed model for survival and by a general linear mixed model for fecundity. The numbers of plant replicates expressing the aphid genes are shown above the bars (n), which are the sums of two independent experiments. Asterisks indicate a significant difference between the effector- and the enhanced green fluorescent protein (eGFP)-expressing peas (***P < 0.001). The dotted lines separate the data that were analyzed independently.

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<table><caption>Fig. 2. Protein alignment of Ap4 orthologs in different aphid species. Protein sequences retrieved from the genome sequences of *Acyrthosiphon pisum* (NP_001156075), *Macrosiphum euphorbiae* (CAI6366837), *Myzus persicae* (XP_022171789), *Rhopalosiphum padi* (Rpa14644.t1), *Rhopalosiphum maidis* (XP_026817788), *Melanaphis sacchari* (XP_025202014), *Pentalonia nigronervosa* (g25341.t1), *Aphis glycines* (g10812.p1), *Myzus cerasi* (Mca07176.t1), *Aphis craccivora* (KAF0770906), and *Aphis gossypii* (XP_027846924) were aligned by ClustalW. The predicted signal peptide sequences are marked with a red rectangle.</caption><thead><tr><th>Signal peptides</th><th></th></tr></thead><tbody><tr><td>Acyrthosiphon pisum</td><td>MAVFKLECFATSVLLELOLADAAGGSKKDPSTDDEASSDGDGEQKPTLFKNIDKIKDS</td></tr><tr><td>Macrosiphum euphorbiae</td><td>MAVFKLECFSTSVLLELOLADAASSKESPSTG-KESNDDGEEQKPTFLKTIDKVKS</td></tr><tr><td>Myzus persicae</td><td>MAVFKLEFCATSVLLELOLADAAGTEKTPKDD-TEPKSEDGEEHTAPFLKTIGKVTDSD</td></tr><tr><td>Rhopalosiphum padi</td><td>MAOTFKLEYFATYTELLOLADSAPQNSPSKSD-TTLNGDEEKEKATLFKNIDNVVDS</td></tr><tr><td>Rhopalosiphum maidis</td><td>MAOTFKLEYFSTYTELLOLADSAPQNSPNKSD-TTLNGDGEKEKATFFKNIDNVVNS</td></tr><tr><td>Melanaphis sacchari</td><td>MTDAFK-FYSVSTTELLOLITDSTTQNYPKSD-TNSKDDSGEEKATLFKNIDKVIES</td></tr><tr><td>Pentalonia nigronervosa</td><td>MTDFFKLECLASTTELLOLTEADPKKSSKD-ELNGDKNYKAPFFKNVDKVNN</td></tr><tr><td>Aphis glycines</td><td>MARALKLECFSTSVLLOLSVNSAPRKSKESDT-DSNADDGEEFKPTLLKNIDRLTKT</td></tr><tr><td>Myzus cerasi</td><td>MWFDIKDMVVKDRLSEVELOLADAAGGSEKTPKN-EPKYDGEEQTAPFLKTLSNVTVNG</td></tr><tr><td>Aphis craccivora</td><td>MIFCKFYKDNILDT-DSNGDGEETPTFLKNIDRLTSS</td></tr><tr><td>Aphis gossypii</td><td>0</td></tr><tr><td>Acyrthosiphon pisum</td><td>KPVDVAKEITSIAKEHLDTFSKDEESTLKAEDEKKQKNVDDEDSIVPKVIONNVVLPAKAF</td></tr><tr><td>Macrosiphum euphorbiae</td><td>RPVKIAKEISSAAEHIGKEFGKDFASALKVEFKQKNVDDENSVPKVIONNVVLPAKV</td></tr><tr><td>Myzus persicae</td><td>RPVRMVKOAATGIAADLKSVTKKFEESNLEESKKQV-VNDENSIIPNFIRNNLIOPGKAA</td></tr><tr><td>Rhopalosiphum padi</td><td>RPIKIVRHLSSNAAKERLNKFSNDEQTILNEDSKNT-VNGENSLIPNAITNNLILPAKAL</td></tr><tr><td>Rhopalosiphum maidis</td><td>RPIKIVROLSNARERLNKFSNDEQTILNEDSKNT-VNGENSLIPNAIONNLILPAKAL</td></tr><tr><td>Melanaphis sacchari</td><td>RPIDMVKKLSNIAEQFNSFSKEEHNTLKDSSKKQV-VNDENSLIPNAVGNNLILPAKAI</td></tr><tr><td>Pentalonia nigronervosa</td><td>KVFEMARKTSEVGGKDLKTFSNNEVNTLNEESRKQV-VFDDNSIT-NTLRNNFYLPAKVF</td></tr><tr><td>Aphis glycines</td><td>RAGKMAROFKNMAKDKASKLSKEFKNSLVEDSKKH-VNGENDLIPNGLRNNVLLPITAA</td></tr><tr><td>Myzus cerasi</td><td>RAKLLRKATDDIAADLKSVTKKFEESNLAESKKQV-VNDKNSLIPNFLLKNNVIDPSKAA</td></tr><tr><td>Aphis craccivora</td><td>RAGKMAROFNTIARDKASKLTDEFKNTLIEDSKKQV-VNGENDFLPNSLKNNVFLPLKAA</td></tr><tr><td>Aphis gossypii</td><td>FVQK--------LGENDLIPNGLRNNVLLPITAA 26</td></tr><tr><td>Acyrthosiphon pisum</td><td>AOTSINTLREPVGNMISNVODTYADELAKSQDENVYPIIGSFTKRANKEVALLKASKDT</td></tr><tr><td>Macrosiphum euphorbiae</td><td>AOTSINALREPVGNTLSSVONEFTNEIAKSEKDEDVYPIIGTFTORANKEVALLKASKNT</td></tr><tr><td>Myzus persicae</td><td>AQTGIDILAEPEVKIVSNIKDTYKDELAKEKEESYVPVIGSYTKIANKEVAILRASKNT</td></tr><tr><td>Rhopalosiphum padi</td><td>VQTSFKTVREPVGEMSSIKKTYDDEIVKSDOEEDVYPIIGAFTKRANKSVAIKASKDT</td></tr><tr><td>Rhopalosiphum maidis</td><td>VQTSFKTVKEPVGEMSSIKKTYDDEIVKSDOEEDVYPIIGAYTKSANKEVAIIRASKDT</td></tr><tr><td>Melanaphis sacchari</td><td>VQTSFKTIGKPTEEM-SNIKKTYDDEIVKSDOEDEGVPIIGAFTKNANKEVAIKASKVV</td></tr><tr><td>Pentalonia nigronervosa</td><td>GOTSINTLKEPEVEETISNIKNTFTEDEFDKAK-KDYAPKIESLIIKKANKEIAVLEALKKT</td></tr><tr><td>Aphis glycines</td><td>AOAGLKTFGEPAVEMMSNVKKKFDEELEKADODENVYPIISAYTKKINKEVALVRASRHT</td></tr><tr><td>Myzus cerasi</td><td>AKTGIDIVGEPIKKTVSDIKERYEGELAKSEKEENYVPIIFGSYTKVVNKPLAIFKASKDV</td></tr><tr><td>Aphis craccivora</td><td>AOVGLKTILGEPEVEETMSNAKKTFDEEIEIRADENYVPIIGAFTKKINKEVALVKASRNT</td></tr><tr><td>Aphis gossypii</td><td>AOASLKTFGEPAVEMMSNVKKKFDEELEKADODENVYPIISAYTKKINKEVALVKASRHT</td></tr><tr><td>Acyrthosiphon pisum</td><td>VGNTEFG-SIFGKASSLWNSSTSSNK-KKKAEKPEKEDK</td></tr><tr><td>Macrosiphum euphorbiae</td><td>VGKTEFG-SIFGKASSLWSSSTSSGK-KNKAEKPEKED</td></tr><tr><td>Myzus persicae</td><td>VGSTLG-SMFGRAKSFWGSS-KKDSDTS</td></tr><tr><td>Rhopalosiphum padi</td><td>VENAIG-GIFGRASSLWNEGTTNT-AEKSKKDK</td></tr><tr><td>Rhopalosiphum maidis</td><td>VENAIG-SIFERASSLWNEGTTNT-AEKSKKDK</td></tr><tr><td>Melanaphis sacchari</td><td>VENAIG-GIFRSVSSIWNEGKKNTEKSVFFSCMVKTIAEESIKTTNEDIKIK</td></tr><tr><td>Pentalonia nigronervosa</td><td>PSKMWSNGKKDKVKKPDEKSEKKK-EEEEDEEEDEEDEK</td></tr><tr><td>Aphis glycines</td><td>TGDAME-GTQKTASTITWNEGKDE-EKTTNEDTKI</td></tr><tr><td>Myzus cerasi</td><td>VGGTFG-SMFGRAKSFWGSS-KEDSGIIYI</td></tr><tr><td>Aphis craccivora</td><td>FGDAMG-GLOKTASTITWNEGKDE-EDN</td></tr><tr><td>Aphis gossypii</td><td>TGDAME-GMOKTASTITWYYGDKGTKTKKNKKDKNNIKNKNKNDDTNK</td></tr></tbody></table>
the library construction, and the interaction of seven of the en-
coded proteins with Ap4 was examined in yeast (the full-length
cDNA of *Psat5g096240* was cloned but could not be introduced
into yeast cells). Yeast cells expressing *Ap4* with *Psat3g196400*
or *Psat2g123160* grew on the selective media lacking histidine
and produced blue pigment on the media containing X-α-Gal,
suggesting their physical interaction in the yeast (Fig. 3). Yeast
cells expressing *Psat2g123160* and *Ap4* grew on the stricter
selective media lacking both histidine and adenine (Fig. 3B),
whereas the cells expressing *Psat3g196400* and *Ap4* did not
(Fig. 3A), indicating a stronger interaction between *Ap4* and
*Psat2g123160* compared with *Psat3g196400*. The expression
of full-length fusion proteins in the yeast cells was supported
by western blotting (Supplementary Fig. S2). No other full-
length proteins interacted with Ap4 or showed autoactivation in
yeast cells. *Psat3g196400* and *Psat2g123160* are the orthologs
of Arabidopsis *AtBPL6* (*At3g01210*) and *AtBPL3* (*At1g14340*),
respectively (Supplementary Fig. S3). The BPA1 (Binding Part-
ner of ACD11)-Like protein (BPL) family is widely conserved
in plants and fungi and shown to be involved in plant immu-
nity (Zhang et al. 2020). Therefore, we named *Psat3g196400*
and *Psat2g123160* as *PsBPL1* and *PsBPL2*, respectively. In-
terProScan predicted that both PsBPL1 and PsBPL2 contain an
RRM_1 (RNA Recognition Motif 1, pfam00076) domain, which
is a conserved feature of the BPL protein family, but it was not
present in all the fragments identified by the Y2H screen (Sup-

plementary Fig. S3; Supplementary Table S4), indicating that
the domain is not essential for the interaction with Ap4. Six
more homologs of PsBPL1 and PsBPL2 were identified from
the *P. sativum* version 1A predicted protein database, and the se-
quences of PsBPL1 and 2 were the closest to each other among
the identified homologs (Supplementary Fig. S3). The other six
homologs that have lower homology with PsBPL1 and 2 were
not identified in the Y2H screening.

Ap4 forms cytosolic puncta and interacts with PsBPL
proteins in planta

To investigate where Ap4 and PsBPL proteins may interact in plant cells, we examined the subcellular localization of Ap4 or PsBPL proteins in *N. benthamiana* epidermal cells by expressing Ap4 fused to synthetic GFP (sGFP) and PsBPL1 or 2 fused to a monomeric red fluorescent protein (mRFP). The fecundity of ArPo58 was significantly increased on the pea expressing the N-terminal fusion of Ap4 with sGFP (sGFP-Ap4) but not by the C-terminal fusion of Ap4 with sGFP (Ap4-sGFP), indicating that the N-terminal fusion with sGFP did not interfere with the function of Ap4 (Supplementary Fig. S4; Supplementary Data). Therefore, sGFP-Ap4 was used to examine its subcellular localization. Unlike sGFP alone, which localized in both the cytosol and nucleus (Supplementary Fig. S5A), sGFP-Ap4 protein localized to large puncta (around 5 µm) in the cytosol (Supplementary Fig. S5B). The mRFP alone showed nuclear-

<table><caption>A</caption>
  <thead>
    <tr>
      <th>pGBK</th>
      <th>pGAD</th>
    </tr>
  </thead>
  <tbody>
    <tr>
      <td>-</td>
      <td>-</td>
    </tr>
    <tr>
      <td>SAP11</td>
      <td>TCP13</td>
    </tr>
    <tr>
      <td>Ap4</td>
      <td>-</td>
    </tr>
    <tr>
      <td>-</td>
      <td><i>Psat3g196400</i><br>(<i>PsBPL1</i>)</td>
    </tr>
    <tr>
      <td>Ap4</td>
      <td><i>Psat3g196400</i><br>(<i>PsBPL1</i>)</td>
    </tr>
  </tbody>
</table>

<figure><img src="image_5.png" /><figcaption>B</figcaption></figure>

<table>
  <thead>
    <tr>
      <th>pGBK</th>
      <th>pGAD</th>
    </tr>
  </thead>
  <tbody>
    <tr>
      <td>-</td>
      <td>-</td>
    </tr>
    <tr>
      <td>SAP11</td>
      <td>TCP13</td>
    </tr>
    <tr>
      <td>Ap4</td>
      <td>-</td>
    </tr>
    <tr>
      <td>-</td>
      <td><i>Psat2g123160</i><br>(<i>PsBPL2</i>)</td>
    </tr>
    <tr>
      <td>Ap4</td>
      <td><i>Psat2g123160</i><br>(<i>PsBPL2</i>)</td>
    </tr>
  </tbody>
</table>

<figure><img src="image_6.png" /></figure>

Fig. 3. Ap4 interacts with A, PsBPL1 and B, PsBPL2 in yeast. The yeasts carrying the two expression vectors, pDEST-GBKT7 and pDEST-GADT7, were cultured on synthetic dropout media lacking leucine (–L), tryptophan (–W), and/or histidine (–H) with X-α-Gal and/or adenine (–A). SAP11 and TCP13 were used as the positive control of the interaction. A dash (–) indicates the empty vectors used as the negative controls. The medium without leucine and tryptophan (–LW) was the yeast mating control. The media without histidine (–LWH) and without both histidine and adenine (–LWHA) were used to examine the interaction between the two test proteins.
cytoplasmic localization (Supplementary Fig. S5C), but in the *PsBPL1-mRFP*-expressed cells, weak mRFP signals were distributed in the cytosol, and some puncta were observed (Supplementary Fig. S5D). The expression of *PsBPL2-mRFP* showed mRFP signals in both the nuclei and cytoplasm and showed some puncta (Supplementary Fig. S5E). When *sGFP-Ap4* and *PsBPL1-mRFP* (Fig. 4A) or *PsBPL2-mRFP* (Fig. 4B) were co-expressed, some puncta of mRFP signals colocalized with the puncta of sGFP signals. Co-expression of Ap4 and PsBPL1 or PsBPL2 did not show a clear influence on each other's subcellular localization (Fig. 4; Supplementary Figs. S5 and S6). The expression of full-length fusion proteins was supported by western blotting (Supplementary Fig. S7).

Interactions of Ap4 and PsBPLs in planta were further analyzed by BiFC. A nematode effector protein, MiEFF1, and its target, Arabidopsis AtUSP, were used as the BiFC positive control, and the reconstituted Venus signal was detected in nuclei (Fig. 5A), as reported previously (Truong et al. 2021). *Ap4* fused with the N-terminal fragment of *Venus* fluorescent gene (*Vn*) and the two *PsBPLs* fused with the C-terminal fragment of *Venus* fluorescent gene (*Vc*) were expressed in *N. benthamiana*. Almost no signal was detected when *Ap4* and an empty Vc vector were co-expressed or when the two *PsBPLs* fused with *Vc* were co-expressed with an empty Vn vector (Fig. 5B; Supplementary Fig. S8C and D). When Ap4 and PsBPL1 or PsBPL2 were co-expressed, puncta of the Venus signal were detected in the *N. benthamiana* cells (Fig. 5C and D; Supplementary Fig. S8A and B), indicating that PsBPL1 and PsBPL2 interact with Ap4 in *N. benthamiana* cells. Although PsBPL2-mRFP was observed in nuclei (Supplementary Fig. S5E), when Vn-Ap4 and Vc-PsBPL2 were co-expressed, the puncta of the Venus signal were observed in the vicinity of nuclei but not within nuclei (Supplementary Fig. S8),

indicating that only the fraction of PsBPL2-mRFP located outside of nuclei interacts with Ap4. The expression of full-length fusion proteins was supported by western blotting (Supplementary Fig. S9). A lower quantity of PsBPL1-Vc compared with Vc-PsBPL2 was detected (Supplementary Fig. S9).

## Discussion

In this study, we hypothesized that *A. pisum* salivary genes that are highly expressed in the pea biotype may be particularly important for the pea biotype to feed on pea plants and may interact with pea proteins. We examined the effect of 10 *A. pisum* salivary effector candidates on *A. pisum* performance and identified one gene, named *Ap4*, that increased the fecundity of the *A. pisum* pea biotype on pea. Pea proteins PsBPL1 and PsBPL2 were identified by Y2H screening, and their in planta interactions were confirmed by BiFC experiments.

The homologs of some of the candidate salivary genes tested in this study have been tested in other plant-aphid interactions. *Mp16*, the ortholog of *Ap4*, and *Mp31*, the ortholog of *A. pisum* *LOC100302326* in *M. persicae*, were identified in the EST library of *M. persicae* salivary glands, but ectopic expression of these two genes in *N. benthamiana* did not enhance the fecundity of *M. persicae* on it (Bos et al. 2010). The Sm9723 protein sequence of the grain aphid (*Sitobion miscanthi*) showed the highest similarity to *A. pisum* *LOC1000160421* among its orthologs in other aphid species (Zhang et al. 2022). Overexpression of *Sm9723* suppresses BAX- and INF1-induced cell death and flg22-induced callose deposition in *N. benthamiana*, and silencing of *Sm9723* expression reduces *S. miscanthi* survival and fecundity on wheat, which suggests that Sm9723 promotes *S. miscanthi* fitness by suppressing host plant defense responses

<figure><img src="image_7.png" /><figcaption>Fig. 4. Ap4 and PsBPL1 or PsBPL2 colocalize in *Nicotiana benthamiana*. sGFP-Ap4 was co-expressed with A, PsBPL1-mRFP (*n* = 7) or B, PsBPL2-mRFP (*n* = 17). White scale bars represent 50 µm, and arrows indicate the colocalized signals. Synthetic green fluorescent protein (sGFP) and monomeric red fluorescent protein (mRFP) signals are shown in green and red, respectively, and a bright-field image is shown for each observation. Similar localization patterns were confirmed in at least three different parts of *N. benthamiana* leaves, and only a representative image is shown for each experiment.</figcaption></figure>
(Zhang et al. 2022). In our study, we did not observe a significant difference in aphid performance on the pea leaves expressing *LOC100160421*. These contrasting results may be due to the different methods used to evaluate the functions of Sm9723 and LOC100160421, but it is also possible that LOC100160421 does not function like Sm9723 in pea. Alternatively, the endogenous expression of *LOC100160421* and other effector genes in *A. pisum* may have been sufficient to suppress the target plant defense, and no effect of the exogenously expressed gene could be detected in the fecundity assay. The same hypotheses apply to explain the negative results, in which expression of candi-

date salivary genes did not increase aphid fecundity, although it is also possible that those aphid genes have no contribution to aphid fitness. The presence of hundreds of putative aphid salivary effector genes and their potential functional redundancy (Boulain et al. 2018) make the evaluation of the effect of a single effector candidate a big challenge. Nonetheless, *Ap4* expression increased significantly *A. pisum* fecundity on pea, indicating its contribution to *A. pisum* performance. Presumptive-orthologous genes of *Ap4* have been identified in 11 aphid species belonging to the Aphididae, but not in other insect families, showing that this gene has evolved specifically in this lineage and suggesting

<figure><img src="image_8.png" /><figcaption>Fig. 5. Bimolecular fluorescence complementation (BiFC) assay in *Nicotiana benthamiana*. **A**, MiEFF1 and AtUSP were used as the positive control of the BiFC assay (n = 5). **B**, The Ap4 fused with the N-terminal fragment of Venus fluorescence protein (Vn-Ap4) and empty vector containing the C-terminal fragment of Venus fluorescence protein (Vc) were used as the negative control (n = 23). Vn-Ap4 was co-expressed with **C**, PsBPL1 (n = 12) or **D**, PsBPL2 (n = 32) fused with Vc in the C and N terminals, respectively. White scale bars represent 50 µm, and arrows indicate the Venus signal. The 6-diamidino-2-phenylindole (DAPI) and Venus signals in the BiFC assay are shown in blue and yellow, respectively.</figcaption></figure>
that it is important for the Aphidinae as it is conserved across
this subfamily.

We identified PsBPL1 and PsBPL2 as Ap4-interacting pea
proteins by Y2H screening and confirmed their interactions in
yeast and in *N. benthamiana* using BiFC. Co-expression of
tagged Ap4 and PsBPLs also suggested that they colocalize in
some puncta formed in *N. benthamiana* cells. Arabidopsis BPA1
and BPL proteins bind to ACD11 (Arabidopsis *accelerated cell
death11*), stabilizing it and regulating reactive oxygen species
(ROS) homeostasis in plants (Li et al. 2019). The two PsBPL
orthologous proteins in Arabidopsis are AtBPL6 (for PsBPL1)
and AtBPL3 (for PsBPL2). The *atbpl6* and *atbpl3* mutants ac-
cumulate more ROS upon flg22 and elf18 treatments and reduce
*Pseudomonas syringae* growth compared with the wild type (Li
et al. 2019). Whether PsBPL1 and PsBPL2 function like AtBPL6
and AtBPL3 and are involved in pea immunity is unknown.

Ap4 is not the only effector that interacts with plant BPL
proteins. It was shown that a *Phytophthora capsici* effector,
RxLR207, which is involved in the transition from biotrophic
to necrotrophic stage of the pathogen, targets Arabidopsis BPA1
and BPL protein family members and disrupts ACD11 stabi-
lization to induce ROS-mediated cell death (Li et al. 2019). Re-
cent advances in effector research revealed that pathogen effec-
tors may target the same host plant proteins. For example, the
SGT1-RAR1-HSP90 forms a chaperone complex, which is in-
volved in diverse biological processes such as the regulation of
immune protein stability in angiosperms (Shirasu 2009), is tar-
geted by several unrelated effectors of microbial pathogens, such
as *Ustilago maydis* (SEE1), *Xanthomonas campestris* (AvrBsT),
*P. syringae* (AvrB), and *Phytophthora sojae* (CRN108) (Carella
et al. 2018). Furthermore, the *M. persicae* effector Mp64 and
*P. capsici* effector CRN83_152 target the same plant immune
regulator, E3 SUMO ligase SIZ1, which confers susceptibility
to *M. persicae* and *P. capsici* in Arabidopsis, implying that con-
vergence of virulence strategies extends to aphid effectors (Liu
et al. 2022). Hence, the fact that *A. pisum* effector Ap4 inter-
acts with the same plant protein group as the *P. capsici* effector
suggests the important and possibly conserved function of BPL
proteins in plant immunity. However, it is still unclear how the
Ap4-PsBPL interaction contributes to the increase in aphid fit-
ness. Subcellular localization analysis showed that Ap4 itself
forms large puncta in the cytoplasm and also near cell borders.
This structure may serve as a place where Ap4 interacts with the
PsBPLs and exerts its function. Ap4 did not accumulate in plant
nuclei, and the BiFC experiment revealed that PsBPL2, which
exhibits nuclear-cytoplasmic localization, interacted with Ap4
outside the nuclei. The protein sequence of Ap4 does not con-
tain a known targeting sequence for transport, except the signal
peptide, and further analyses of the Ap4-localized structure may
provide a hint to understand the function of Ap4. In yeast cells,
comparable amounts of PsBPL1 and PsBPL2 were detected by
the western blots, but in *N. benthamiana* cells, significantly less
PsBPL1 compared with PsBPL2 was detected, which may be
due to different expression levels but also due to the specific
subcellular localization of these proteins in plant cells.

The Ap4 protein is detected in pea aphid salivary glands of
the alfalfa biotype (Carolan et al. 2011; Boulain et al. 2018), but
its expression level in the pea biotype is more than four times
higher than in the alfalfa biotype on average (Boulain et al. 2019).
Although Ap4 expression in pea leaves increased the fecundity
of pea biotype ArPo58, it did not promote the survival of the
alfalfa biotype, LL01, on pea. Therefore, Ap4 alone may not be
enough for LL01 to feed on the pea plant, which is a non-host
for the alfalfa biotype, and other salivary effectors of the pea
biotype may be required for the alfalfa biotype to overcome the
defense system of pea. In addition, the expression level of the
gene in salivary glands may not be corelated with the amount of

the protein secreted into the plant. Examining whether Ap4 also
promotes the alfalfa biotype to feed on alfalfa (*Medicago sativa*)
and whether it interacts with alfalfa PsBPLs will deepen our
understanding of the host specialization processes of *A. pisum*
biotypes.

In conclusion, the transient expression of the salivary effec-
tor *Ap4* gene promotes the fecundity of the pea biotype on the
pea plant. The identification of its plant targets, PsBPL1 and
PsBPL2, suggests the involvement of BPL proteins in pea im-
munity against aphids and provides another example of the con-
vergence of effector targets. Further analyses of PsBPL1 and
PsBPL2 functions will be one of the next challenges to under-
stand the pea immune system against aphids.

**Materials and Methods**

Plants and aphids

Vicia faba (genotype Castel) was used for aphid rearing and grown in a growth chamber set at 18°C with a 16-h day/8-h night photoperiod. *P. sativum* (genotype AeD99OSW-50-2-5) (Ollivier et al. 2022) and *N. benthamiana* were grown in a growth chamber set at 21°C with a 16-h day/8-h night photoperiod and used for the aphid performance assay and confocal experiment, respectively. Two-week-old *P. sativum* plants at the two-pair leaf stage and 4-week-old *N. benthamiana* plants at the eight-leaf stage were used for the agroinfiltration assay.

Two different clones of *A. pisum*, ArPo58 of the pea bio-
type and LL01 of the alfalfa biotype, both lacking known aphid
secondary bacterial symbionts, were used for aphid perfor-
mance assays (Boulain et al. 2019). Both clones were reared on
*V. faba*, which is a universal host of the various *A. pisum* biotypes
(Peccoud and Simon 2010).

Plasmid construction and Ap4 protein sequence analysis

To clone the 10 selected effector candidate genes, heads of the
pea biotype clone P123 were used for RNA extraction with the
NucleoSpin RNA XS kit (Macherey-Nagel) and cDNA synthe-
sis with SuperScript III First-Strand Synthesis System (Invitro-
gen). The coding sequences of the genes without a signal peptide
region were PCR amplified by Phusion high-fidelity DNA poly-
merase (Thermo Fisher) with gene-specific primers that contain
partial sequences of the attB1 and attB2 Gateway recombina-
tion sites (Supplementary Table S5). The fragments were further
amplified with attB1 and attB2 adapter primers and cloned into
pDONR207 with Gateway BP Clonase II Enzyme Mix (Invitro-
gen) to create Gateway entry clones. Full-length pea genes were
cloned in the same way from pea cDNA used in the Y2H analy-
sis. These Gateway entry clones were used for cloning the genes
into destination plasmids for gene expression. The cloned DNA
sequences were verified by Sanger sequencing. The sequence of
the protein encoded by *Ap4* of the clone P123 was confirmed
to be the same as that of the clone LL01 (NCBI SRA accession
SRX20811676).

Sequences of Ap4 protein (NP_001156075) orthologs
were identified from the NCBI website and available aphid
genomes on the AphidBase website (https://bipaa.genouest.org/
is/aphidbase) (Legeai et al. 2010) by BlastP with an e-value
cutoff of 1e-5. Multiple alignment of these proteins was made
with the ClustalW function in Geneious software (version 7.1)
(Thompson et al. 1994). Signal peptide regions were predicted
by SignalP 5.0 (Almagro Armenteros et al. 2019).

Agroinfiltration

The AGL1 strain of *Agrobacterium tumefaciens* was used to
transiently express genes in *P. sativum* and *N. benthamiana*
leaves (Guy et al. 2016). Overnight cultured cells were sus-
pended in the induction buffer to an OD₆₀₀ (optical density at
600 nm) of 0.5. Cells were infiltrated into leaves of 2-week-old *P. sativum* (genotype AeD99OSW-50-2-5) 3 days before installation of aphids or 4-week-old *N. benthamiana* 2 days before the observation by confocal microscopy.

## Aphid performance assays

The candidate aphid effector genes were cloned in a pEAQ-HT-DEST1 plasmid by Gateway LR reaction (Invitrogen) (Sainsbury et al. 2009), and *Ap4* was also cloned in pGWB405 and pGWB406 plasmids (Nakagawa et al. 2007) to create sGFP-fused *Ap4* in C-terminal (*Ap4*-sGFP) and N-terminal fusion (*sGFP*-Ap4), respectively. The fecundity assay was conducted as described in Guy et al. (2016). AGL1 cells transformed with these plasmids were infiltrated in pea leaves 3 days before aphid installation, and AGL1 cells carrying an eGFP-expressing plasmid were used as the control. Adult aphids were installed on *V. faba* 24 h before starting the assay to produce age-synchronized nymphs. For the performance assay of the pea biotype, six ArPo58 nymphs (1 day old) were confined in a clip cage on an agroinfiltrated leaf of *P. sativum*. The number of surviving ArPo58 was counted on the seventh day, then one medium-sized ArPo58 adult was transferred to a new *P. sativum* leaf, which had been infiltrated with the same *Agrobacterium* 3 days before. On days 11 and 14 post-aphid installation, the clip cages were opened, and the nymphs in the cages were counted and removed. The sum of these two counts (total number of offspring produced by a single aphid during 14 days after installation) was used as a proxy of aphid fecundity. For the performance assay of the alfalfa biotype, clone LL01 was used. Six LL01 nymphs (1 day old) were installed in the same way as ArPo58, and then the surviving LL01 were counted on day 7. Because LL01 cannot survive on pea for 14 days, only the survival assay was performed. Each infiltrated leaf was counted as one replicate, and at least 14 replicates were performed for each examined gene from at least two independent experiments.

## Y2H analysis

To generate a pea cDNA library, the pea biotype clone (P123), the alfalfa biotype clone (LSR1), and the clover biotype clone (YR2) were used to infest three pea genotypes: an *A. pisum*-resistant genotype (AeD99OSW-50-2-5), a susceptible genotype (CE101 = FP) (Ollivier et al. 2022), and the genotype used for whole-genome sequencing (CAMEOR) (Kreplak et al. 2019). The cDNA library was created using the pool of RNA isolated from the three pea genotypes with or without aphid infestation. Two-week-old peas were infested with 200 one-day-old nymphs of the pea-adapted biotype (pea biotype P123) or the non-pea-adapted biotypes (mixture of 100 alfalfa biotype LSR1 and 100 clover biotype YR2), and the aerial parts of the pea plants were harvested 24 and 72 h after infestation and flash frozen. Aphids were carefully removed from the plants floating on liquid nitrogen, and noninfested peas were harvested at the same time. Total RNA was extracted from the collected peas (leaves and stems) using the NucleoSpin RNA Plant kit (Macherey-Nagel), and then an equal quantity of RNA extracted from each condition was mixed and sent to Hybrigenics Services.

The Y2H screening was performed by Hybrigenics Services, S.A.S., Evry, France (http://www.hybrigenics-services.com). The total RNA from *P. sativum* leaves and stems (1,215 µg) was used for the extraction of messenger RNA (GenElute mRNA Miniprep Kit; Sigma). The polyA+ RNA was used for the synthesis of random-primed cDNA (SuperScript Double-Stranded cDNA Synthesis; Invitrogen), resulting in an average of 700 nucleotides of cDNA fragments. The cDNA fragments were cloned into a pP6 prey vector and used to produce the *P. sativum* leaf and stem library in yeast (PSLS_RP), which is expected to contain 10 million independent fragments. The coding sequence for

*Ap4* (aa 24-214) was PCR-amplified and cloned into pB66 as a C-terminal fusion to the Gal4 DNA-binding domain (Gal4-Ap4). The construct was used as a bait to screen the *P. sativum* leaf and stem library. pB66 derives from the original pAS2ΔΔ vector (Fromont-Racine et al. 1997), and pP6 is based on the pGADGH plasmid (Bartel et al. 1993). For the N-Gal4-Ap4-C construct, 29 million (2.8-fold the complexity of the library) clones were screened using the mating approach with YHGX13 and CG1945 yeast strains, and 194 colonies were selected on a medium lacking tryptophan, leucine, and histidine and supplemented with 0.5 mM 3-aminotriazole to handle bait autoactivation. The prey fragments of the positive clones were amplified by PCR and sequenced at their 5' and 3' junctions. The resulting sequences were used to identify the corresponding interacting proteins in the GenBank database (NCBI) using a fully automated procedure, and predicted biological scores (Fromont-Racine et al. 1997) were assigned.

To confirm the physical interactions between *Ap4* and the pea proteins identified by the Y2H screening, the genes encoding full-length proteins were PCR-amplified using the primers designed based on the genome sequence of *P. sativum* v1a (Kreplak et al. 2019) and the pea cDNA library and cloned into the Gateway entry vector pDONOR207 with the primers listed in Supplementary Table S6. *Ap4* was cloned into a Gateway-compatible pDEST-GBK7T7 vector to fuse to the DNA-binding domain of GAL4, and pea genes were cloned into pDEST-GADT7 to fuse to the activation domain of GAL4 (Rossignol et al. 2007). The clones containing aster yellows phytoplasma effector SAP11 and Arabidopsis TCP13 were used as the positive control of the interaction (Sugio et al. 2014). pDEST-GBK7T7-Ap4 and pDEST-GADT7 pea proteins were transformed into *Saccharomyces cerevisiae* strains Y187 and AH109, respectively (Matchmaker III; Clontech Laboratories). The interactions of *Ap4* and pea proteins were confirmed by mating the two yeast strains and examining their growth in synthetic dropout media depleted of leucine, tryptophan, histidine, and adenine. To confirm the activity of α-galactosidase induced by the interactions, a final concentration of 40 µg/ml of X-α-Gal (Takara Bio) was added to the media.

## PsBPL protein identification and sequence analysis

The PsBPL1, PsBPL2, and published AtBPL proteins were used as the queries to identify homologous BPL proteins in pea by BlastP against the *P. sativum* version 1A protein database with an e-value cutoff of 1e-5 (Kreplak et al. 2019). Six additional homologs of PsBPL1 and PsBPL2 were identified. The phylogenetic analysis of the total eight PsBPL and six AtBPL proteins was performed with the neighbor-joining method in MEGA 11 (Tamura et al. 2021). The protein sequences of the pea BPLs were aligned by ClustalW.

## BiFC and confocal microscopy

The *Ap4* gene without the sequence encoding its signal peptide was cloned into pGWB406 (N-terminal fusion with sGFP, sGFP-Ap4), and *PsPBL1* and *PsBPL2* were cloned into pGWB454 (C-terminal fusion with mRFP, PsBPL-mRFP) by Gateway LR reaction. To study the in vivo interactions of Ap4 and PsBPLs, these genes were cloned in BiFC vectors containing split Venus fluorescence proteins, pDEST-GW-VYNE (C-terminal fusion for MiEFF1, MiEFF1-Vn), pDEST-VYNE(R)-GW (N-terminal fusion for Ap4, Vn-Ap4), pDEST-GW-VYCE (C-terminal fusion for PsBPL1, PsBPL1-Vc), and pDEST-VYCE(R)-GW (N-terminal fusion for AtUSP and PsBPL2, Vc-AtUSP, and Vc-PsBPL2) (Gehl et al. 2009). *Meloidogyne incognita* MiEFF1 and Arabidopsis AtUSP were used as the BiFC positive control (Truong et al. 2021). Those constructs were transformed into AGL1 cells. The fused proteins were expressed in *N. benthami-
ana leaves by agroinfiltration. Expression of fluorescent proteins in pea leaves did not give a clear image as in *N. benthamiana*; therefore, *N. benthamiana* was used for all the studies to observe fluorescent proteins. Two days after infiltration, the fluorescence signals were acquired with a Leica SP8 laser scanning confocal microscope equipped with a 40× or 20× objective and a laser module (excitation at 405, 488, and 561 nm). To visualize nuclei, 1 to 10 µg ml⁻¹ of 4, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) suspended in phosphate-buffered saline buffer was infiltrated into the leaves 2 h before the observation. Observation of an area distant from the other observations was counted as one replicate, and at least six replicates using more than two plants were performed for each experiment. At least two independent experiments were conducted on different days.

## Western blotting

To confirm expression of fusion proteins in yeast, 10 ml of overnight liquid culture of yeast cells in leucine and tryptophan dropout media was collected by centrifugation and incubated in 600 µl of 0.1 M NaOH for 6 min, spun down, resuspended in 50 µl of 4× NuPAGE LDS sample buffer (Invitrogen), and incubated at 95°C for 5 min, and 10 µl was subjected to western blot analysis.

Two leaf discs (diameter = 2 cm) of *N. benthamiana*-expressing fusion proteins were flash frozen in liquid nitrogen and crushed in a buffer containing 6 M urea, 2 M thiourea, and protease Inhibitor (Pierce Protease Inhibitor; Thermo Fisher) and incubated at 95°C for 10 min. The debris was spun down, and the supernatant was mixed with 4× NuPAGE LDS sample buffer (Invitrogen) and incubated at 95°C for 5 min, and 16 µl of the samples was subjected to western blot.

The samples were separated in Any kD Mini-PROTEAN TGX Precast Protein Gel (Bio-Rad) and transferred to 0.45-µm Immobilon-P PVDF membranes (Millipore) using the semi-dry blotting system. The western blots were incubated with an appropriate primary antibody. Depending on the experiments, mouse monoclonal anti-HA antibody (Invitrogen 26183), anti-c-Myc antibody (Invitrogen MA1-980), rabbit poly clonal anti-GFP (Thermo Fisher A11122), and anti-RFP (Thermo Fisher 600-401-379) antibodies were used as the primary antibodies, and then peroxidase-conjugated goat anti-rabbit (Bio-Rad 1662408) or mouse IgG (Merck AP130P) was used as the secondary antibody. Detection of bound antibodies was conducted with Immobilon Western Chemiluminescent HRP Substrate (Millipore) or SuperSignal West Atto Ultimate Sensitivity Chemiluminescent Substrate (Thermo Scientific).

## Statistical analyses

All statistical analyses were conducted in R version 4.1.2 (R Core Team 2021). As each effector gene was studied in the aphid performance assay twice, the combined results of the two independent experiments were analyzed. Therefore, we included in our statistical modeling the period ID as a random factor to consider time dependency in the analysis. The effect of the salivary effector gene on aphid survival rate was analyzed by using a generalized linear mixed model considering a binomial error and a logit link function. The influence of the salivary effector gene on the aphid fecundity was tested by using a general linear mixed model. General model assumptions were verified by checking residual normality and homoscedasticity by plotting residuals versus fitted values. In the case of assumption deviation, the effect of the salivary effector gene on aphid fecundity was analyzed using a generalized linear mixed model, assuming a Poisson error and a log link function. The significance of the tested fixed factor in generalized linear models was tested using a likelihood ratio test, and its significance in the general models was estimated using a Fisher test. In all models, the models'

coefficients were analyzed in case of significance of the effector gene effect to detect which salivary effector genes differed from the control (eGFP).

## Acknowledgments

We thank Marie-Laure Pilet-Nayel, Isabelle Glory, and Angélique Lesné for providing the pea seeds; Fabrice Legeai for his support in the bioinformatic analyses; Bruno Favery for providing *MiEFFI* and *AtUSP* clones for the BiFC experiment; Saskia Hogenhout for providing SAP11 and TCP13; Tsuyoshi Nakagawa for providing pGWB vectors; Jörg Kudla for providing BiFC vectors; and Stéphanie Dutertre of the Microscopy Rennes Imaging Center (MRic, BIOSIT, Biogenouest) for assistance. MRic is member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04). We are grateful to Sam Mugford and Qun Liu for discussions on the western blotting methods and to the editor and the anonymous reviewers for their constructive feedback and valuable suggestions, which significantly contributed to the improvement of this manuscript.

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