# Facilitation of symplastic effector protein mobility by paired effectors is conserved in different classes of fungal pathogens

Nacera Talbi, Mila Blekemolen, Slavica Janevska, Daniel Zendler, Herman van Tilbeurgh, Isabelle Fudal, Frank Takken

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## ► To cite this version:

Nacera Talbi, Mila Blekemolen, Slavica Janevska, Daniel Zendler, Herman van Tilbeurgh, et al. Facilitation of symplastic effector protein mobility by paired effectors is conserved in different classes of fungal pathogens. Molecular Plant-Microbe Interactions, 2024, 37 (3), pp.304-314. 10.1094/MPMI-07-23-0103-FI. hal-04232770

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Distributed under a Creative Commons Attribution 4.0 International License
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Facilitation of symplastic effector protein mobility by paired effectors is conserved in different classes of fungal pathogens.

Nacera Talbi *¹, Mila C. Blekemolen*², Slavica Janevska², Daniel Zendler², Herman van Tilbeurgh³, Isabelle Fudal^¹ & Frank L.W. Takken^².

*equal contribution, ^equal contribution

**Affiliation**

1 Université Paris-Saclay, INRAE, UR BIOGER, 91120 Palaiseau, France

2 Molecular Plant Pathology, Swammerdam Institute of Life Science (SILS), University of Amsterdam, Amsterdam, the Netherlands.

3 Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France

**Corresponding authors:**

Frank Takken, Email address: [F.L.W.Takken@uva.nl](mailto:F.L.W.Takken@uva.nl)

and

Isabelle Fudal, Email address: [Isabelle.Fudal@inrae.fr](mailto:Isabelle.Fudal@inrae.fr)

**Author's ORCID ID**

Mila Blekemolen: 0000-0001-8646-4129

Nacera Talbi: 0000-0002-8676-8066

Herman van Tilbeurgh: 0000-0002-6723-1036

Slavica Janevska: 0000-0001-7361-495X

Daniel Zendler: 0000-0002-4951-7663

Isabelle Fudal: 0000-0002-9105-9432

Frank Takken: 0000-0003-2655-3108

Total Wordcount: 6419

Wordcount sections: Summary (191), Introduction (1036), Material & Methods (1405), Results (1940), Discussion (1450)

Number of main figures: 6 (All colour figures)

Number of supplemental figures: 7

Number of supplemental tables: 5

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**Summary**

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Recently, it was uncovered that plant pathogens produce effectors that spread via plasmodesmata to allow modulation of host processes in distal uninfected cells. *Fusarium oxysporum* forma specialis *lycopersici* (*Fol*) facilitates effector translocation by expansion of the size exclusion limit of plasmodesmata using the Six5/Avr2 effector pair. How other fungal pathogens manipulate plasmodesmata is unknown. We recently reported that many fungal pathogens belonging to different families carry effector pairs that resemble the *SIX5/AVR2* gene pair from *Fol*. Here, we performed structural predictions of three of these effector pairs from *Leptosphaeria maculans* (*Lm*) and tested their ability to manipulate plasmodesmata and to complement the virulence defect of a *Fol SIX5* knockout mutant. We show that the AvrLm10A homologs are structurally related to FolSix5 and localize at plasmodesmata when they are expressed with their paired effector. Furthermore, these effectors were found to complement FolSix5 function in cell-to-cell mobility assays and in fungal virulence. We conclude that distantly related fungal species rely on structurally related paired effector proteins to manipulate plasmodesmata and facilitate effector mobility. The wide distribution of these effector pairs implies Six5-mediated effector translocation to be a conserved propensity among fungal plant pathogens.

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Key words (5-8): Plasmodesmata, cell-to-cell mobility, plant pathogens, fungi, *Fusarium*, *Leptosphaeria*, effector pairs, 3D structure.

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**Introduction**

During infection plant pathogens secrete effectors, which are key elements of pathogenesis modulating host immunity and facilitating infection (Lo Presti et al., 2015; Rocafort et al., 2020). Some effectors function extracellularly (i.e., in the apoplast), while others exert their functions inside plant cells. Some pathogens have developed systems for the delivery of their effectors into plant cells, e.g., (i) the type III secretion system of bacteria, (ii) stylet injection following glandular effector production of nematodes, or (iii) production of effectors directly inside the plant cells for viruses (Cunnac et al., 2009; Eves-van den Akker & Birch, 2016; Ray & Casteel, 2022). Phytopathogenic filamentous fungi secrete their effectors first in the apoplastic space or in the extra-haustorial space surrounding the feeding structure penetrating the plant cell wall (Rocafort et al., 2020). Some of these effectors can be taken up and act inside host cells, but the uptake mechanism for these effectors remains enigmatic (Petre & Kamoun, 2014). Many intracellular-acting effectors from fungi have been discovered (Figueroa et al., 2021; He et al., 2020), including many for *Fusarium oxysporum* (Tintor et al., 2020).

Some intracellular effectors have been shown to be mobile: they move via the symplast allowing modulation of host processes ahead of the infection zone (Blekemolen et al., 2022; Cao et al., 2018; Djamei et al., 2011; Khang et al., 2010; Sheshukova et al., 2020). Plasmodesmata (PD), tightly regulated pores connecting neighbouring cells, facilitate intracellular communication between plant cells and movement of effectors. The size exclusion limit (SEL) of PD determines whether a protein can pass the membrane-spanning channel. Viruses are known to manipulate PD through their movement proteins (MP), increasing the SEL and allowing for transport of larger molecules. Recently, other pathogens have also been reported to manipulate PD during infection. The bacterium *Pseudomonas syringae* secretes HopO1-1 to modulate PD function and to promote host colonization (Aung et al., 2019). Besides HopO1-1, also other *P. syringae* effector proteins were reported to be mobile and translocated through PD (Li et al., 2021). The Cmu1 effector from the maize smut fungus *Ustilago maydis* was proposed to move to neighbouring cells through PD (Djamei et al., 2011). The effector proteins PWL2 and BAS1 from the rice blast fungus *Magnaporthe oryzae* move into uninfected cells following their delivery into infected rice cells (Khang et al., 2010). These mobile effectors have been hypothesized to act as “conditioners” for pathogenic infection (Blekemolen et al., 2022; Sheshukova et al., 2020; Cao et al., 2018; Djamei et al., 2011; Khang et al., 2010;). By inducing favourable conditions ahead of the infection front, e.g., through PTI (pathogen-associated molecular pattern (PAMP)-triggered immunity) suppression, they promote colonization (Couto & Zipfel, 2016). Whether symplastic mobility of fungal effectors is a widespread phenomenon, and if so, which factors are involved in effector mobility, is currently unknown.
How fungal effectors manipulate PD has been best studied for the Avr2(Six3)/Six5 effector pair of the tomato-infecting fungal pathogen *F. oxysporum* forma specialis (f. sp.) *lycopersici* (*Fol*) (Blekemolen et al., 2022; Cao et al., 2018). The Avr2(Six3)/Six5 effector pair functions as a fungal MP: Six5 interacts with Avr2 (Six3) specifically at PD and together they manipulate the SEL of PD to facilitate cell-to-cell movement of Avr2(Six3) and other cytosolic *Fol* effectors (Blekemolen et al., 2022). As Six3 alone can trigger I-2-mediated resistance in heterologous systems, it was renamed to Avr2 (Houterman et al., 2009), although both Avr2 and Six5 are required for I-2-mediated resistance in tomato (Ma et al., 2015). *AVR2* and *SIX5* are organized as neighbouring genes in opposite transcriptional orientation, and their expression is controlled by a shared promoter region (Ma et al., 2015; Schmidt et al., 2013). Both genes are expressed early during the infection process upon colonization of the vasculature of the plant by the fungus (Ma et al., 2015). Avr2 and Six5 are required for full virulence of the fungus and either gene knockout results in a strain that is less pathogenic on tomato (Houterman et al., 2009; Ma et al., 2015). The observation that Avr2 transgenic plants no longer exhibit immune responses upon exposure to various PAMPs such as flg22, chitin, chitosan, nlp23 and SCOOPs is in agreement with the recent demonstration that Avr2 targets PTI signalling (Blekemolen et al., 2023; Coleman et al., 2021; de Lamo et al., 2021; Di et al., 2017; Tintor et al., 2020). The crystal structure of Avr2 has been determined, revealing a β-sandwich structure with two antiparallel β-sheets resembling the fold of the ToxA protein from *Pyrenophora tritici-repentis* (Di et al., 2017). Whereas Avr2 has at least two activities, PTI suppression and PD manipulation, the main role of the less-well-studied Six5 appears to act in conjunction with Avr2 to increase the SEL of PD (Blekemolen et al., 2022). Whether other fungal pathogens use a similar strategy to manipulate their host is currently unknown.

Homologs of *SIX5* were identified in 30 plant pathogenic fungi of the Dothidomycete, Sordariomycete and Leotiomycete families, including *Leptosphaeria maculans*, the causal agent of oilseed rape stem canker, part of them being associated with an effector partner (Talbi et al., 2023). One of these homologs, AvrLm10A, physically interacts with the *L. maculans* effector AvrLm10B and both genes are required to trigger *Rlm10* resistance (Petit-Houdenot et al., 2019). Resembling *Fol* Avr2 and Six5, AvrLm10A and AvrLm10B are encoded by two neighbouring genes that are organized in head-to-head orientation. Recently, four paralogs of the AvrLm10A/AvrLm10B pair were identified in the *L. maculans* genome. All four gene pairs share a similar opposite transcriptional orientation and are co-expressed during infection. A physical interaction between the encoded protein pairs could be demonstrated for one of them, AvrLm10A_like2/AvrLm10B_like2 (Talbi et al., 2023).

The observed similarities between the *F. oxysporum* and *L. maculans* effector pairs raise the question whether they exert the same functionality, i.e., SEL expansion through PD manipulation facilitating increased effector mobility. Here, three *L. maculans* AvrLm10A/AvrLm10B-like effector pairs, all highly expressed during oilseed rape infection, were structurally and functionally characterized by
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studying (i) the predicted structural conservation between Avr2- and Six5-like effectors of *F. oxysporum* and *L. maculans*, (ii) the localization of the paired *L. maculans* effectors at the PD, (iii) the ability of *LmSix5* homologs to replace the role of Six5 in PD manipulating activity and virulence of *F. oxysporum*.

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## Materials & Methods

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### Protein structure analysis.

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The 3D structure predictions of the different proteins were generated using AlphaFold2 v1.5.2 with default parameters (https://github.com/sokrypton/ColabFold, Mirdita et al., 2022) using the protein sequences of the effectors without their signal peptide (Petit-Houdenot et al., 2019; Talbi et al. 2023).

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The highest ranked models were chosen for comparison. Structure comparison and structure-based protein sequence alignments were performed using the Chimera software, version 1.16 (Pettersen et al., 2004).

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### Vector construction for effector localization, cell-to-cell movement assays and complementation constructs.

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Binary vectors (2xFP (FP5149); 35S:ΔspAvr2 (FP3436); 35S:ΔspSix5 (FP3430); 35S:GUS (FP3426)) used for cell-to-cell movement assays with 2xFP (fusion of a GFP and NeonGreen (NG) fluorophore) have been described (Blekemolen et al., 2018, 2022).

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For localization and detection of cell-to-cell movement of *L. maculans* effectors, RNA of cotyledons and stems of the susceptible oilseed rape cultivar Darmor infected by the reference strain *L. maculans* v23.1.3 (7 dpi and 28 dpi, respectively) was extracted using the TRIzol® Reagent (Invitrogen, Cergy Pontoise, France) and cDNA was generated using oligo-dT-primed reverse transcription with PrimeScript Reverse Transcriptase (Clontech, Palo Alto, CA, USA) as described (Plissonneau et al., 2016). The genes of interest were amplified using specific primers (Table S3) for gateway cloning from cDNA isolated from oilseed rape cotyledons infected with *L. maculans* at 7 dpi or from cDNA of oilseed rape stem infected with *L. maculans* at 28 dpi using Phusion polymerase (Invitrogen, Carlsbad, USA).

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PCR products flanked by attB recombination sites were cloned into the pDONR207 vector (Invitrogen, Carlsbad, CA, USA) via a BP (recombines an attB with an attP site) reaction to obtain entry vectors carrying attL sequences according to the supplier's recommendations (Thermofisher, Waltham, MA, USA). The inserts cloned into the entry vectors were subsequently recombined into pSite-dest-GFP or pK2GW7 destination vectors (Chakrabarty et al., 2007; Karimi et al., 2002) via an LR reaction (recombines attL in an attR site), to generate pK2GW7:AvrLm10A (FP8030); pK2GW7:AvrLm10B (FP8031); pK2GW7:AvrLm10A_like1 (FP8032); pK2GW7:AvrLm10B_like1 (FP8033);

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pK2GW7:AvrLm10A_like2 (FP8034) pK2GW7:AvrLm10B_like2 (FP8035); and
pSite:AvrLm10A:GFP; pSite:AvrLm10A_like1:GFP; pSite:AvrLm10A_like2:GFP.

For complementation assays, vector assembly was performed using yeast recombination cloning
making use of ca. 25 bp overhangs to facilitate seamless cloning (Colot et al., 2006). First, a base vector
was generated from the yeast shuttle vector pNDH-OGG (Schumacher, 2012), which allows for *in locus*
integration of a gene of interest at the constitutively expressed DNA damage response gene of *Fol*, the
*DDR48/FOXG_02247* locus (Janevska et al., 2017). Upstream (including *DDR48*) and downstream
sequences of *DDR48* were amplified from *Fol* gDNA as template using primers FP9236/FP9237 and
FP10355/FP9239 (Table S3), respectively. As an insert, *PoliC:GFP:Tgluc* was amplified with
FP10185/FP9242 from pNDH-OGG, which allows for constitutive expression of *GFP* via the *oliC*
promoter from *Aspergillus nidulans* (Schumacher, 2012). For nourseothricin selection of *Fol*
transformants, *natR* including its promoter *PtrpC* was amplified with FP9021/FP9909 from pZPnat1
(GenBank AY631958.1). *Saccharomyces cerevisiae* BY4741 (Euroscarf) was transformed with the
obtained fragments as well as with SacII/PmeI-digested pNDH-OGG, yielding the base vector pDDN-
OGG. In the next step, *PoliC:GFP* of pDDN-OGG was exchanged for the *SIX5* homologs, in each case
including the native *Fol SIX5* promoter (Prom) (Ma et al., 2015) and signal peptide-encoding sequences.
Therefore, *Prom:SPsix5* was amplified with FP10860/FP10861 (Table S3) from *Fol* gDNA, and the
inserts *SIX5* (FP1993/FP10862), *AvrLm10A* (FP10863/FP10864), *AvrLm10A_like1*
(FP10865/FP10866) and *AvrLm10A_like2* (FP10867/FP10868) were amplified from cDNA. S.
*cerevisiae* BY4741 was transformed with *Prom:SPsix5* and the respective inserts, together with the
linearized vector backbone (FP9021/FP10354), yielding the (cross-)complementation vectors
pDDN:*SIX5<sup>C</sup>*, pDDN:*AvrLm10A<sup>CC</sup>*>, pDDN:*AvrLm10A_like1<sup>CC</sup>* and pDDN:*AvrLm10A_like2<sup>CC</sup>*. All
expression vectors were verified by sequencing.

Agrobacterium tumefaciens-mediated transient transformation of Nicotiana benthamiana leaves.

For detection of cell-to-cell movement, *A. tumefaciens* infiltration (agro-infiltration) of four week-old *N. benthamiana* plants was performed as described (Ma et al., 2012). Co-infiltrations of *A. tumefaciens* GV3101 (pMP90) strains harbouring binary vectors encoding *2xFP/mCherry:HDEL* together with strains harbouring *ΔspAVR2*, *ΔspSIX5*, *AvrLm10B*, *AvrLm10A*, *AvrLm10B_like1*, *AvrLm10A_like1*, *AvrLm10B_like2*, *AvrLm10A_like2* or *GUS* were carried out at an OD<sub>600 nm</sub> of 0.5 for each strain. The viral silencing suppressor gene *p19* was co-infiltrated along with the other constructs at an OD<sub>600 nm</sub> of 0.1 (Cao et al., 2018). Leaves were subjected to confocal microscopy 72 h post infiltration. *N. benthamiana* plants were grown, prior to infiltration, in a climate-controlled greenhouse at 22 °C, with 70% relative humidity, and under short daylight conditions: 13 h dark, 11 h light.

Determination of a potential plasmodesmatal (co)localization of *L. maculans* effectors was performed
as described (Petit-Houdenot et al., 2019). Co-infiltrations of *A. tumefaciens* GV3101 (pMP90) strains
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carrying a binary vector with gene constructs AvrLm10A:GFP, AvrLm10A_like1:GFP or AvrLm10A_like2:GFP, mCherry:PDCB1 (Grangeon et al., 2013) and AvrLm10B, AvrLm10B_like1 or AvrLm10B_like2 were carried out at an OD₆₀₀ nm of 0.5 each. *N. benthamiana* was cultivated, prior to infiltration, in a growth chamber with a 16 h day (25 °C, 50% humidity) and 8 h night (22 °C, 50% humidity) regime. Leaves were subjected to confocal microscopy 48 and 72 h post infiltration.

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Confocal microscopy.

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For cell-to-cell movement assays, confocal microscopy was performed using a Nikon A1 microscope (Nikon Instruments Inc, Melville, NY, USA). Excitation of GFP and Neongreen fluorophores (Blekemolen et al., 2022) was carried out at 488 nm with an Ar-ion laser and the emitted signal was selected using a 500-525/550 nm bandpass filter. mCherry was excited at 561 nm with a dpss (diode-pumped solid state) laser and light signal emitted was selected with a 570/595-620 nm bandpass filter. The detector gain was between 800 and 900. Multi-labelling with mCherry and GFP or Neongreen was imaged by sequential scanning to monitor co-localization of the fluorescent proteins. To determine cell-to-cell movement events, the number of green-fluorescent cells flanking a transformed area were counted and border length was measured. The number of cell-to-cell movements was normalised to a 750 µM border. A Mann-Whitney *U* test was applied on the number of cell-to-cell movement events per border using the software PRISM 7.0 (GraphPad).

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For localization assays of *L. maculans* effectors proteins at PD, microscopy analyses were performed using a Leica TCS SPE laser scanning confocal microscope and a 63X oil objective lens. *N. benthamiana* leaves were observed 48 h and 72 h post infiltration. The following excitation and emission wavelengths were used; GFP: excitation 488 nm, emission captured using a 496-546 nm broad-pass filter; mCherry excitation 532 nm, emission 578 to 649 nm. The detector gain was between 800 and 900. All images are representative of at least twenty scans.

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Fusarium *protoplast* transformation and analysis of transformants.

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The *Fol* 007ΔSix5 deletion strain, in which the *SIX5* gene (*FOXG_22493*) is replaced with a hygromycin cassette, was described previously (Ma et al., 2015). Protoplasts of *Fol* 007 Δsix5 were transformed as described (Tudzynski et al., 1999). Linear (cross-)complementation constructs were transformed, after amplification with primers FP9236/FP9239 (Table S3). Transformants were selected on 150 µg/mL nourseothricin (Jena Bioscience, Jena, Germany) and transferred to potato dextrose agar (PDA, BD Difco; Sigma Aldrich, St. Louis, MO, USA) using double selection, *i.e.*, 100 µg/mL hygromycin B (Sigma Aldrich, St. Louis, MO, USA) and 150 µg/mL nourseothricin. *In locus* integration of *SIX5* homologs was verified *via* absence of untransformed nuclei, using *DDR48* diagnostic primers

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FP9240/FP9241 (PCR WT), and presence of inserts using primers FP9615/FP9071 (PCR *Prom_SIX5* – *Tgluc*). This yielded at least 2 independent transformants each for *SIX5C*, *AvrLm10ACC*, *AvrLm10A_like1CC* and *AvrLm10A_like2CC*.

Fusarium *infection assay*.

The tomato (*Solanum lycopersicum*) cultivar C32 that is susceptible to *Fol* 007 (race 2) was used for infection assays (Kroon & Elgersma, 1993). Tomato plants were grown in a climate-controlled greenhouse at 25 °C with 65% relative humidity and a 16 h light/8 h dark regime. 250 mL flasks with 100 mL NO₃ medium (0.17% yeast nitrogen base without amino acids or ammonium sulphate, 3% sucrose and 100 mM KNO₃), inoculated with mycelium from PDA plates, were shaken at 25 °C (125 rpm, for 3-5 days) to produce spores. Infection of tomato seedling (15-20 per treatment) with the *Fol* strains was performed by trimming the roots (1 cm remaining) and dipping them for two minutes into a fungal spore suspension (10⁷ spores/mL) as described (Di et al., 2016). Three weeks post inoculation, Fresh Weight (FW) and Disease Index (DI) were scored as described (Gawehns et al., 2014), adding a score of DI = 5 when plants were dead. As FW and DI were scored with different scales, the datasets were standardized for a later scatter plot using the integrated R function “scale” (R Core Team, 2021). Clustering of the data was performed using mclust v5.4.10 according to the supplied documentation (https://cran.r-project.org/web/packages/mclust/vignettes/mclust.html) (Scrucca et al., 2016).

## Results

*The AvrLm10A/AvrLm10B pair, their homologs in L. maculans and the Six5/Avr2 pair from F. oxysporum share structural similarities.*

Since *F. oxysporum* effector Six5 and *L. maculans* effectors AvrLm10A, AvrLm10A_like1 and AvrLm10A_like2 share between 36 and 52 % amino acid sequence identity (Talbi et al., 2023), we hypothesized that these effectors could possess a similar protein fold. Using Alphafold2, 3D structural models of the effectors (without their predicted signal peptides) were generated. Models with high confidence indices were generated (pLDDT > 90, over the whole sequence length; Supplementary Table S1). Notably, all four effector proteins were predicted to share the same fold, consisting of a twisted four stranded β-sheet covered by two helices on one side and a β-hairpin and long loop on the other side (Fig. 1A-B). The models for Six5, AvrLm10A, AvrLm10A_like1 and AvrLm10A_like2 superpose with RMSD (root-mean-square deviation) values between 0.8 and 1.1 Å after superposition (Supplementary Table S2). Seven cysteines are conserved and form three disulphide bridges and one remaining unpaired cysteine (Fig. S1)
275 AvrLm10B has low protein sequence identity with AvrLm10B_like1 and AvrLm10B_like2 (27% and 24% respectively; Talbi et al., 2023) but lacks detectable sequence identity with *F. oxysporum* effector Avr2. Alphafold2 generated a good model for AvrLm10B (pLDDT 85), a lower-quality model for AvrLm10B_like1 (pLDDT 65), and a low-confidence model for AvrLm10B_like2 (pLDDT 34; Fig. S2). Although the N-terminal regions (approximately 50 amino acids) adopt different conformations, the models for AvrLm10B and AvrLm10B_like1 display the same β-sandwich fold (Fig. 2A). The model for AvrLm10B_like2 is also composed of two sheets but their orientation and topology diverge from that of the two other family members (Fig. 2A). We then compared these models with the crystal structure of Avr2 (pdb accession number 5OD4) that also adopts a β-sandwich-type of fold consisting of two anti-parallel β-sheets (Di et al., 2017). This type of fold has previously been found for the *P. tritici-repentis* protein ToxA. As can be seen in Fig. 2B, the AvrLm10B model superposes onto the crystal structure of Avr2 with an RMSD of 1.9Å, illustrating their similarity. A detailed table with all the superposition statistics is provided (Supplementary Table S2). Taken together, the *L. maculans* and *F. oxysporum* effectors do not only resemble each other in being encoded by gene pairs possibly sharing a promoter region, but also the protein models reveal similar folds.

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291 The *L. maculans* effectors AvrLm10A and AvrLm10A_like2 localize at plasmodesmata in the presence 292 of AvrLm10B or AvrLm10B_like2. 293

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294 The structural similarity between the *L. maculans* AvrLm10A pairs and the *F. oxysporum* Six5/Avr2 pair 295 suggests a potential conservation of function. Six5 and Avr2 physically interact and, *in planta*, co- 296 localize at plasmodesmata (PD) (Cao et al., 2018). Recently, a physical interaction was also reported 297 for AvrLm10A/AvrLm10B and AvrLm10A_like2/AvrLm10B_like2 but not for 298 AvrLm10A_like1/AvrLm10B_like1 (Petit-Houdenot et al., 2019; Talbi et al., 2023). Interaction of the 299 Avr2 and Six5 effectors with PD results in enlargement of their SEL, facilitating transport of Avr2 and 300 other Six effectors and proteins to adjacent cells (Blekemolen et al., 2022; Cao et al., 2018). To 301 determine whether the identified Six5 homologs also localize at PD, the effectors AvrLm10A, 302 AvrLm10A_like1 and AvrLm10A_like2 were fused to GFP. AvrLm10A-GFP, AvrLm10A_like1-GFP 303 and AvrLm10A_like2-GFP proteins were produced together with the mCherry-labelled PD marker 304 protein PDCB1 (plasmodesmata callose-binding protein 1) in *N. benthamiana* (Fig. S3). PDCB1-mCherry accumulation results in weak red fluorescence in the cytosol and a strong signal at the PD, 305 which is visible as punctate spots at the plasma membrane (Fig. S3, second column). The AvrLm10- 306 GFP proteins accumulate in cytoplasm and nucleus, but no (or very little) localization was observed at 307 the plasmodesmata. Next the AvrLm10-GFP fusions were transiently co-expressed with their non- 308 tagged effector partner in *N. benthamiana*. Co-expression of the AvrLm10A:GFP/AvrLm10B or 309 AvrLm10A_like2:GFP/AvrLm10B_like2 pairs resulted, besides green fluorescence in the nucleus and 310
cytosol, also in bright signals at the PD (Fig. 3). The co-localization of the GFP and mCherry signal, visible as yellow dots in the merged panels, suggest that AvrLm10A and AvrLm10A_like2 localize at PD when produced with their effector partner (Fig.3, first panel). Whereas expression of AvrLm10A_like1:GFP resulted in green-fluorescent cytoplasm and nuclei of transformed cells, no distinct spots co-localizing with the PD were detected for the AvrLm10A_like1:GFP/AvrLm10B_like1 pair (Fig. S4). Immunoblot analysis with anti-GFP antibodies confirmed the presence of intact recombinant proteins (Talbi et al., 2023). Similar as for the *F. oxysporum* Six5/Avr2 pair, the presented data suggest that AvrLm10A/AvrLm10B and AvrLm10A_like2/AvrLm10B_like2 localize at PD.

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320 L. maculans *effector pairs promote cell-to-cell movement of a 53 kDa fluorescent protein by altering the SEL of PD*

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323 Given the PD localization of two of the *L. maculans* effector pairs, we set out to determine whether these effectors alter the SEL of these structures using cell-to-cell translocation assays of 2x Fluorescent Protein (2xFP), consisting of a GFP-NeonGreen (NG) fusion protein. The size of the 2xFP protein (53 kDa) exceeds the SEL of PD, thereby restricting its ability to translocate to neighbouring cells. When the SEL is increased, e.g., due to the action of PD-manipulating effectors, the 2xFP protein becomes hypermobile (Blekemolen et al., 2022). *A. tumefaciens* strains carrying a binary vector encoding either mCherry-HDEL and 2xFP or the effector pairs AvrLm10B/AvrLm10A, AvrLm10B_like1/AvrLm10A_like1, or AvrLm10B_like2/AvrLm10A_like2 were co-infiltrated in *N. benthamiana* leaves. The *F. oxysporum* SIX5/AVR2 pair served as a positive control for 2xFP hypermobility. All proteins were produced without their native signal peptides to assure their cytosolic location. The mCherry-HDEL protein was used to identify transformed cells, as the HDEL endoplasmatic reticulum (ER) retention signal tethers the red fluorescent protein to the ER, confining the protein to the transformed cell. To determine the basal mobility of the 2xFP protein, a GUS-encoding construct was used as control. In the GUS control, green and red fluorescence were readily detectable in the agro-infiltrated tissue, but green fluorescence was rarely observed in the untransformed cells neighbouring the infiltrated area (Fig. 4; Fig. S5A). Confinement of the 2xFP protein to the infiltrated region agrees with the inability of the fusion protein to translocate to adjacent cells via PD. In the presence of Avr2 and Six5, however, green fluorescence was observed in the cytoplasm and/or nucleus of cells bordering a transformed region (marked by arrowheads, Fig. S5B), indicating cell-to-cell movement of 2xFP (Fig 4). Interestingly, like Six5/Avr2, expression of the *L. maculans* effector pairs AvrLm10B_like1/AvrLm10A_like1 and AvrLm10B_like2/AvrLm10A_like2 resulted in an increase of cell-to-cell movement of 2xFP as compared to the GUS control (Fig. 4, Fig. S5C-D). The AvrLm10B/AvrLm10A pair did not promote movement of 2xFP. We conclude that two *L. maculans* effector pairs can promote cell-to-cell movement of 2xFP, suggesting a function of these effectors in extending the SEL of PD.

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Complementation of the cell-to-cell movement activity of effector pair Six5/Avr2 by L. maculans effectors

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The activity of two *L. maculans* effector pairs on PD, together with the structural similarity between *F. oxysporum* Six5-Avr2 and the *L. maculans* AvrLm10A-AvrLm10B homologs suggests that the individual functions of the effectors could be conserved as well. Production of either Avr2 or Six5 alone is not sufficient to change PD conductivity, showing that the combined action is required to alter the SEL (Cao et al., 2018). To investigate this further, we tested whether the *L. maculans* AvrLm10 effectors (AvrLm10A, AvrLm10A_like1, AvrLm10A_like2; AvrLm10B, AvrLm10B_like1, AvrLm10B_like2) could functionally complement either Six5 or Avr2 in a cell-to-cell translocation assay. Thereto, *A. tumefaciens* carrying a binary vector encoding both mCherry-HDEL and 2xFP was co-infiltrated in *N. benthamiana* leaves with strains encoding either i) GUS, or ii) Avr2 together with Six5, AvrLm10A, AvrLm10A_like1 or AvrLm10A_like2, or iii) Six5 together with Avr2, AvrLm10B, AvrLm10B_like1 or AvrLm10B_like2. As expected, no cell-to-cell movement of 2xFP outside the infiltrated area was observed in the GUS control (Fig. 5; Fig. S5A). In the presence of Six5/Avr2, the 2xFP showed hypermobility and translocated from cell to cell (Fig. 5), as shown by the green fluorescence in the cytoplasm and/or nucleus (marked by arrowheads) of untransformed cells bordering a transformed leaf region (Fig. S5B). Notably, the combination of Avr2 with each of the three *L. maculans* Six5 homologs also resulted in increased cell-to-cell movement of 2xFP (Fig. 5; Fig. S5F-H). The number of translocation events per infiltrated area for the *LmSix5* homologs was like that of Six5, showing that the *L. maculans* proteins can complement the *F. oxysporum* Six5 function in the presence of Avr2. Pairing Six5 with the *L. maculans* Avr2 homologs, AvrLm10B_like1 and AvrLm10B_like2 also promoted cell-to-cell movement of 2xFP, whereas AvrLm10B did not complement Avr2 activity (Fig. 5; Fig. S5I-K). In conclusion, the three *L. maculans* Six5 homologs and two Avr2 homologs (AvrLm10B_like1 and AvrLm10B_like2) can complement the PD-manipulating activity of the *F. oxysporum* Six5/Avr2 effector pair. The observed cross-complementation in the translocation assay highly suggests a common functionality of these effectors in manipulating PD.

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L. maculans Six5 homologs complement the virulence defect of SIX5 deletion in Fol

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Since the three *L. maculans* Six5 homologs complemented the PD-manipulating activity of Six5 in conjunction with Avr2, we tested whether they could also restore virulence of a *Fol* SIX5 deletion strain. SIX5 deletion leads to virulence defects corresponding to a reduction of disease symptom development following inoculation of tomato (Ma et al., 2015). To test whether the *L. maculans* Six5 homologs can functionally complement Six5 function, protoplasts of *Fol* 007 ΔSix5 were transformed with constructs encoding *FolSIX5*, or *L. maculans* effector genes *AvrLm10A*, *AvrLm10A_like1* or *AvrLm10A_like2*. The transgene cassettes were inserted into a constitutively expressed locus (*DDR48*, *FOXG_02247*) to

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minimize differences in transgene expression levels due to ectopic integrations at different loci (Janevska et al., 2017). Expression of the genes was controlled by the *Fol SIX5* promoter and each open reading frame was fused to the native *Fol SIX5* signal peptide-encoding sequence (Fig. S6). Disease assays were performed by dipping 10-day-old tomato seedlings in spore suspensions of the *Fol* 007 wildtype (WT), *Fol* 007 ΔSix5 mutant or the *SIX5C*, *AvrLm10ACC*, *AvrLm10B_like1CC* and *AvrLm10A_like2CC* (cross)-complementation strains. Two independent transformants (T) per construct were included in the disease assays. Three weeks after inoculation, disease symptoms were scored, disease index indicating an increase in disease severity ranging from 0 to 5 (with 0 corresponding to no symptoms and 5 to dead plants, Gawehns et al., 2014) and plant fresh weight was measured (Fig. S7). Cluster modelling using mclust revealed four distinct clusters within our dataset (Scrucca et al., 2016). One cluster contains both mocks and encompasses groups that have a disease index of 0 in combination with a high plant weight (Fig. 6; cluster 4 (pink)). Inoculation of the seedlings with the WT *Fol* 007 caused typical *Fusarium* wilt symptoms, such as wilting, stunting, and vessel browning. Both WT *Fol* 007 inoculation groups as well as *AvrLm10A_like2CC* T2 (I) are found in cluster 3 (purple) (Fig. 6) based on their low weight and high disease index. The *Fol* ΔSix5 infected plants localize together with *AvrLm10A_like1CC* T6 (I) and T9 (II) in cluster 2 (orange)). These plants showed reduced disease symptoms as well as increased fresh weight of the plants as compared to plants inoculated with the WT, consistent with the reduced virulence of the *SIX5* deletion strain (Fig. 6). Since *AvrLm10A_like1CC* T6 (I) and T9 (II) group in the same cluster, this implies that the *AvrLm10A_like1* gene does not complement the virulence defect caused by deletion of *SIX5*. Transformation of native *SIX5* into the *FolΔSix5* mutant background resulted in a higher disease index as compared to seedlings inoculated with the *Fol* ΔSix5 strain. The *SIX5C* complementation strains group together in cluster 1 (green) with transformants carrying *AvrLm10A* or *AvrLm10A_like2* (and one transformant carrying *AvrLm10A_like1*). Taken together, these data indicate that, except for *AvrLm10A_like1*, the *SIX5* homologs complement the virulence defect of the *Fol* ΔSix5 mutant to the same extent as native *SIX5*.

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412 Discussion

413

414 In this study, we showed that three *L. maculans* AvrLm10 effector pairs
415 (i) have similar predicted structural folds as Avr2 and Six5 from *Fol*, (ii) permit 2xFP protein
416 translocation, possibly by increase the SEL of PD, and (iii) (partially) cross-complement the virulence
417 defect caused by *SIX5* deletion in *Fol*. These results support our hypothesis that the ability to
418 symplastically transport effector proteins is conserved at least in two fungal pathogens from distinct
419 classes (Dothideomycetes and Sordariomycetes), but also raise questions about the involvement of
420 structurally-related effector families for infection mechanisms and in the potential induction of broad-
421 spectrum resistance.
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422

423 The ability to symplastically transport effector proteins is important for the virulence of several plant
424 pathogens (Aung et al., 2020; Cao et al., 2018; Khang et al., 2010; Liu et al., 2021). In *Fol*, the
425 Six5/Avr2 effector pair facilitates effector mobility through PD manipulation and the inactivation of
426 *SIX5* and/or Avr2 leads to virulence defects (Houterman et al., 2009; Ma et al., 2015; Cao et al., 2018).
427 Here, we investigated the ability of the *L. maculans* AvrLm10 effector pairs to promote 2xFP movement
428 and of AvrLm10A(-like) to associate to PD. While the results obtained for AvrLm10A_like2 and
429 AvrLm10B_like2 clearly demonstrated their ability to allow cell-to-cell movement and of
430 AvrLm10A_like2 to associate to PD in the presence of AvrLm10B_like2, results for the other *L.
431 maculans* effector pairs were more unambiguous. Indeed, AvrLm10A_like1 did not localize at PD in
432 the presence of AvrLm10B_like1, but AvrLm10A_like1 and AvrLm10B_like1 promoted cell-to-cell
433 movement of 2xFP, while AvrLm10A localized at PD in the presence of AvrLm10B but AvrLm10A
434 and AvrLm10B did not promote cell-to-cell movement of 2xFP. Of note, the lack of PD localization of
435 the AvrLm10A_like1 effector in presence of AvrLm10B_like1 is not due to poor accumulation of the
436 effector-GFP-fusion protein, since it was readily detected on protein blots at similar intensities as the
437 other fusion proteins (Talbi et al., 2023). Talbi et al. (2023) did not observe physical interaction between
438 AvrLm10A_like1 and AvrLm10B_like1 and whether these paired *L. maculans* effectors interact
439 specifically at PD in presence of additional protein(s) remains to be determined. The
440 AvrLm10A/AvrLm10B pair on the contrary, did not facilitate cell-to-cell movement of 2xFP, even
441 though this pair did co-localize at PD. A possible explanation could be the weak physical interaction
442 between AvrLm10A and AvrLm10B (Petit-Houdenot et al., 2019; Talbi et al., 2023) as AvrLm10A
443 could complement the PD manipulation activity of Six5/Avr2 suggesting that AvrLm10A might form
444 a more stable interaction with Avr2.
445

445

446 Seventy-one Six5/AvrLm10A family members have been identified in over 30 different species of
447 Dothideomycetes, Sordariomycetes, and Leotiomycete phytopathogenic fungi (Talbi et al., 2023), most
448 Six5/AvrLm10A family members being present in *F. oxysporum* and *Colletotrichum* species. Some
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fungal species were found to carry up to five Six5/AvrLm10A homologs per genome (Talbi et al., 2023). Over half of the identified *SIX5/AvrLm10A* genes have a putative effector-encoding gene as neighbouring gene. It is tempting to speculate that the analogous effector pairs found in other phytopathogenic fungi have a similar ability to facilitate cell-to-cell movement as the *L. maculans* AvrLm10A/B and *Fol* Six5/Avr2 pairs.

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While fungal effectors have long been described as species-specific, the availability of an increasing number of fungal effector repertoires - and the resolution of their 3D structure - has facilitated the identification of structural analogues among effectors that show low level of sequence identity. This is notably the case for the RALPH (RNAse-Like Proteins Associated with Haustoria), MAX (for Magnaporthe Avrs and ToxB like), ToxA-like, LARS (for Leptosphaeria AviRulence and Suppressing), and FOLD (*Fusarium oxysporum* f.sp. *lycopersici* dual domain) effectors (Di et al., 2017; de Guillen et al., 2015; Lazar et al., 2022; Pedersen et al., 2012; Wang et al., 2007; Yu et al., 2021). Moreover, the growing efficiency of *ab initio* protein structure predictors such as DeepMind's Alpha Fold2 has allowed proteome-wide prediction and classification in fungal effector families, showing the expansion of some families in specific phylogenetic groups while other folds are present across the fungal species (Seong and Krasileva, 2023; Derbyshire and Raffaele, 2023). The same way, despite their low sequence identity, the AvrLm10 family members from *L. maculans* and the distantly related Six5/Avr2 pair from *Fol* were predicted to share similar protein folds. Recently, a combination of structure determination and structural predictions on *Fol* effectors identified several structural effector families (Yu et al., 2021). The *Fol* effectors Six7 and Six8 were classified, together with Avr2, in the ToxA-like effector class. Like the Six5/Avr2 and AvrLm10 pairs, the homologs of Six8 from *Fol* and *F. oxysporum* f. sp. *conglutinans* (*Focn*) form head-to-head oriented gene pairs with neighbouring effector genes, *FoPSL1* (*PSE1-LIKE 1*) and *FocnPSE1* (*PAIR WITH SIX8 1*), respectively (Ayukawa et al., 2021). Notably, the protein models for *FoPSL1* and *FocnPSE1* strongly resemble those of Six5 and AvrLm10A. *SIX7* and *SIX12* also form a pair of neighbouring genes in head-to-head transcriptional orientation. Even though the structural prediction for Six12 had a very low confidence score, not allowing its categorization in a structural family, the conservation of cysteine spacing sequence suggests that it could belong to the Six5 / AvrLm10A family. Altogether, the combination of a ToxA-like (Avr2/AvrLm10B-like) protein and a Six5/ AvrLm10A-like protein seems to be prominent for effector pairs in *F. oxysporum*, *L. maculans* and potentially other fungi from the Dothideomycetes and Sordariomycetes classes.

Conservation in effector structure could indicate similar molecular interactions between the effectors and/or between the effectors and PD localized protein(s), and a conserved activity. The AvrLm10A-SIX5 family, which is specifically expressed during the biotrophic stages of infection, was previously hypothesized to have a role in the hemi-biotrophic lifestyle, by favouring the biotrophy before necrosis development (Talbi et al., 2022). Notably, silencing of *AvrLm10A* revealed a role of this effector in
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restricting leaf lesion development (Petit-Houdenot et al., 2019), thus lengthening the biotrophic stage of infection before the fungus switches to necrotrophy. The main function of Six5 appears to be the manipulation of PD to facilitate movement of the associated effector (Blekemolen et al., 2022), a function that might be shared by Six5 structural homologs. Inactivation of *SIX5* in *Fol* resulted in a virulence defect which was, at least partially, restored by *AvrLm10A*(-like) effector genes. The combination of Avr2 with each of the three *L. maculans* AvrLm10A(-like) effectors also resulted in increased cell-to-cell movement of 2xFP, showing that the *L. maculans* proteins can complement the *F. oxysporum* Six5 function in the presence of Avr2. This observation implies that the AvrLm10A(-like) effectors possess the propensity to be taken up by the plant cells after being secreted by *Fol*, and to function together with Avr2 to manipulate PD. The structural analogy between Avr2 and AvrLm10B(-like) effectors is lower but is suggestive of a common ancestral topology. Two AvrLm10B(-like) effectors complemented the cell-to-cell movement when paired with Six5, indicating compatibility of these effector pair partners despite the large evolutionary distance between *Fol* and *L. maculans* and the low structural analogies between these effectors.

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Both Six5 / Avr2 and AvrLm10A / AvrLm10B trigger recognition by a specific resistance gene, respectively *I-2* from tomato and *Rlm10* from *B. nigra* (Houterman et al., 2009; Ma et al., 2015; Petit-Houdenot et al., 2019). Some structurally related effectors can be recognized by the same resistance gene. For instance, two effectors of the MAX family, AVR-Pia and AVR1-CO39 from *Pyricularia oryzae* are both recognised by the rice RGA4/ RGA5 pair (Cesari et al., 2013; de Guillen et al., 2015). Analogously, variants of the barley MLA recognize structurally related avirulence effectors from *Blumeria graminis*, which belong to the RALPH family (Bauer et al., 2021). Finally, Lazar et al. (2022) found that two effectors from the LARS family, AvrLm3 of *L. maculans* and Ecp11-1 of *Fulvia fulva*, are both recognized by *Rlm3* from oilseed rape, suggesting that *Rlm3* could confer broad-spectrum resistance. *Rlm10* does not appear to recognise members of the *L. maculans* AvrLm10 family outside of AvrLm10A and AvrLm10B as silencing of these genes suffices to escape *Rlm10* recognition (Petit-Houdenot et al., 2019). However, it would be interesting to determine whether *Rlm10* could be a broad spectrum resistance gene, able to recognise other members of the AvrLm10 - Six5/Avr2 family to protect oilseed rape from additional fungal pathogens, including *Fusarium* sp..

Taken together, our findings show that non-haustorium-forming fungi with a complex hemi-biotrophic lifestyle rely on structurally-related effector pairs for symplastic mobility of proteinaceous effectors. PD are highly conserved cellular structures and the finding that phylogenetically distant fungi use similar effector pairs to manipulate symplastic mobility implies a broad conservation of this property among phytopathogenic fungi. It will be interesting to explore the virulence-promoting functions of the effector pairs in other plant pathogenic fungi and to study whether their Avr2-like proteins possess similar PTI-suppressing roles or if they have other virulence-related functions.
523 Acknowledgments

524 We would like to thank Ludek Tikovsky, Harold Lemereis, John-Paul Mony Morris, Gabrielle Bertiner
525 and Guillaume Lucas for plant care, Thomas Aalders, Babette Vlieger and Margarita Šimkovicová for
526 assistance with performing bioassays, Petra Houterman and Bas Beerens for lab management/care,
527 Xiaorong Tao for the pZK538 construct, Joachim Goedhart for the Neongreen construct, Ronald
528 Breedijk, the LCAM microscopy department and Kaori Sakai for assistance with microscopy, Laetitia
529 Dupont for assistance with molecular cloning, all members of the “Effectors and Pathogenesis of L.
530 maculans” group for their advice. Martijn Rep for critical reading and discussion of the manuscript. No
531 conflict of interest is declared.

532

533 Author contributions

534 M.C.B., N.T., S.J., and D.Z. designed and conducted the experiments. M.C.B., N.T., I.F. and F.L.W.T.
535 conceptualized the experiments. M.C.B., N.T., H.v.T., I.F. and F.L.W.T. wrote the paper.

536

537 Funding

538 F.L.W.T. and M.C.B. were supported by the NWO-Earth and Life Sciences funded VICI project no.
539 865.14.003. N.T. was funded by a PhD salary from the University Paris-Saclay. N.T, H.v.T. and I.F.
540 were supported by the French National Research Agency project STARlep (ANR-20-CE20-0026).
541 S.J. was supported by a grant from the German Research Foundation (DFG project no. 408033566).
542 D.Z. was supported by TKI T&U grant LWV20.105. The “Effectors and Pathogenesis of *L. maculans*”
543 group benefits from the support of Saclay Plant Sciences-SPS (ANR-17-EUR-0007).

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545

546 Figure captions

547

548 Fig. 1. 3D models of AvrLm10A, AvrLm10A_like1, AvrLm10A_like2, and Six5.

549 (a) Alphafold2 models of *L. maculans* AvrLm10A, AvrLm10A_like1 and AvrLm10A_like2 (Mirdita
550 et al., 2022). The signal peptide sequence was precluded from the prediction of each effector protein.
551 The Alphafold2 model of Six5 is shown on the right. (b) Superposition of the AvrLm10A and Six5
552 models.

553

554 Fig. 2. 3D models of AvrLm10B, AvrLm10B_like1 and AvrLm10B_like2 and comparison with
555 the structure of Avr2.

556 (a) Alphafold2 models of AvrLm10B, AvrLm10B_like1 and AvrLm10B_like2 (Mirdita et al., 2022).
557 The signal peptide sequence was precluded from the prediction of each effector protein. The crystal
558 structure of Avr2 is shown on the right (pdb # 5OD4, Di et al. 2017). (b) Superposition of the
559 AvrLm10B model and the Avr2 crystal structure.
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**Fig. 3. AvrLm10A and AvrLm10A_like2 relocalize at the plasmodesmata in the presence of their paired effector**

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Confocal microscopy images of agro-infiltrated *N. benthamiana* leaves transiently expressing either *AvrLm10A:GFP* and *AvrLm10B*, or *AvrLm10A_like2:GFP* and *AvrLm10B_like2*. Plasmodesmata were visualized by co-expressing the gene encoding the plasmodesmata-associated plant marker protein PDCB1 fused to mCherry. Arrowheads mark green dots originating from the GFP-tagged effector protein colocalizing with the red fluorescent signal emitted by m-Cherry-labelled PDCB1 at plasmodesmata. Images were recorded 72 h post agro-infiltration and are representative of at least 20 scans. Experiments were repeated three times. Scale bar represents 10 µm.

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**Fig. 4. Two of the three *L. maculans* effector pairs promote cell-to-cell movement of 2xFP**

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Co-infiltration of *A. tumefaciens* strains carrying a binary vector encoding mCherry-HDEL (transformation control) and 2xFP together with strain(s) expressing either *GUS*, *SIX5/AVR2*, *AvrLm10B/AvrLm10A*, *AvrLm10B_like1/AvrLm10A_like1* or *AvrLm10B_like2/AvrLm10A_like2* into *N. benthamiana* leaves. Green and red fluorescence was analysed 72 h post agro-infiltration using confocal microscopy. Cell-to-cell movement of 2xFP (a GFP-NeonGreen fusion protein) in the presence of *Fol SIX5/AVR2* or the *L. maculans* effector pairs was quantified in 15-20 individual images originating from two independent biological replicates. Each datapoint represents a single image of a transformation border where the number of cell-to-cell movement events (i.e., number of cells) were scored. (***, *P*-value < 0.0007, **, *P* < 0.004, *, *P* < 0.02, ns, non-significant, Mann-Whitney *U* test). Error bars represent the standard error of the mean.

**Fig. 5. Two *L. maculans* Six5 homologs and all three Avr2 homologs complement ability of the Six5/Avr2 effector pair to manipulate plasmodesmata allowing cell-to-cell movement of a 53 kDa protein.**

Co-infiltration of *A. tumefaciens* strains carrying a binary vector encoding mCherry-HDEL and 2xFP together with strain(s) expressing either *GUS*, *SIX5/AVR2*, *AVR2/AvrLm10A*, *AVR2/AvrLm10A_like1*, *AVR2/AvrLm10A_like2*, *AvrLm10B/SIX5*, *AvrLm10B_like1/SIX5* or *AvrLm10B_like2/SIX5* into *N. benthamiana* leaves. Green and red fluorescence was analysed 72 h post agro-infiltration using confocal microscopy. Transformed cells were identified based on their red fluorescent ER originating from the mCherry-HDEL protein. Quantification of cell-to-cell movement of 2xFP in presence of the effector pair combinations is based on the analysis of approximately 19-20 individual images originating from two independent biological replicates. Each datapoint represents a single image of a transformation border where the number of cell-to-cell movement events (i.e., number of cells) were scored. (****, *P*-value < 0.0001, **, *P* < 0.008, ns, non-significant, Mann-Whitney *U* test). Error bars represent the standard error of the mean.
<caption>**Fig. 6.** Complementation of virulence of a *F. oxysporum* ΔSix5 deletion strain with *L. maculans* SIX5 homologs.</caption>

Ten-day-old tomato seedlings were inoculated with water (mock), *Fol* 007 (WT), *Fol* 007 ΔSix5, or (cross-)complementation (CC) transformants (*SIX5*<sup>C</sup> T4, T6, T7; *AvrLm10A*<sup>CC</sup> T1, T2; *AvrLm10A_like1*<sup>CC</sup> T1, T6, T9; *AvrLm10A_like2*<sup>CC</sup> T1, T2). Fresh weight (FW) and disease index (DI) were assessed three weeks post inoculation; two individual biological replicates were performed (replicate 1 N=15, replicate 2 N=20 plants per isolate/treatment). Cluster analysis based on standardized average of the mean. FW and DI showed four distinct clusters within the dataset.

**Supplemental information**

<caption>**Supplemental Fig. S1.** Structure-based alignments of the effector protein pair Six5/Avr2 and three *L. maculans* Six5/Avr2 homologs.</caption>

(a) Multiple alignments of Six5 protein sequence and three *L. maculans* Six5 homologs based on their structures using UCSF Chimera software (Pettersen et al., 2004). (b) Multiple alignments of Avr2 protein sequence and three *L. maculans* Avr2 homologs based on their structures using UCSF Chimera software.

<caption>**Supplemental Fig. S2:** Per-residue confidence estimate for the three predicted AvrLm10B homologues.</caption>

The plot illustrates the per-residue pLDDT score across the *Alphafold2* models of AvrLm10B, AvrLm10B_like1 and AvrLm10B_like2.

<caption>**Supplemental Fig. S3:** In absence of their effector partners, AvrLm10A-GFP, AvrLm10A_like1-GFP and AvrLm10A_like2-GFP do not accumulate at plasmodesmata.</caption>

Confocal microscopy images of agro-infiltrated *N. benthamiana* leaves transiently expressing either *AvrLm10A-GFP*, *AvrLm10A_like1-GFP* or *AvrLm10A_like2:GFP*. Plasmodesmata were visualized by co-expressing the gene encoding the plasmodesmata-associated plant marker protein PDCB1 fused to mCherry. Images were recorded 48h post agro-infiltration and are representative of at least 20 scans. Scale bar represents 20 µm.

<caption>**Supplemental Fig. S4.** AvrLm10A_like1/AvrLm10B_like1 do not co-localize at plasmodesmata.</caption>

Confocal microscopy images of agro-infiltrated *N. benthamiana* leaves transiently expressing *AvrLm10A_like1:GFP* and *AvrLm10B_like1*, together with the gene encoding the plasmodesmata-associated plant marker protein, PDCB1 fused to mCherry. Green fluorescence was observed in cytosol
and nucleus of transformed cells, but not in discrete spots co-localizing with the red fluorescent signal emitted by m-Cherry-tagged PDCB1. Images were recorded 72 h post agro-infiltration and are representative of at least 20 scans. Experiments were repeated three times. Scale bar represents 50 µm.

<caption>**Supplemental Fig. S5.** *L. maculans* effector proteins functionally complement the ability of the Six5/Avr2 effector pair to manipulate plasmodesmata allowing cell-to-cell movement.</caption>

(a-k) Confocal images of *N. benthamiana* leaves agro-infiltrated with a binary vector encoding mCherry-HDEL and 2xFP together with a strain(s) expressing either *GUS*, ΔspAVR2/ΔspSIX5, AvrLm10A/AvrLm10B, AvrLm10A_like1/AvrLm10B_like1, AvrLm10A_like2/AvrLm10B_like2, AVR2/AvrLm10A, AVR2/AvrLm10A_like1, AVR2/AvrLm10A_like2, AvrLm10B/SIX5, AvrLm10B_like1/SIX5 or AvrLm10B_like2/SIX5. Green and red fluorescence was analysed 72 h post infiltration using confocal microscopy. Transformed cells show both ER-localized red fluorescence and a nucleo-cytoplasmic-localized 2xFP green-fluorescent signal. Cell-to-cell movement of the green 2xFP protein to untransformed neighbouring cells is indicated by arrowheads. Scale bar represents 50 µm.

<caption>**Supplemental Fig. S6.** Validation of Fol 007 ΔSix5 (cross-)complementation by SIX5 and its homologs.

(a) Vectors for expression of *Fol SIX5* and the homologs from *L. maculans* (*AvrLm10A*, *AvrLm10A_like1*, *AvrLm10A_like2*) fused to the native *Fol SIX5* promoter and signal peptide. (b) *In locus* integration of the constructs at the constitutively expressed *DDR48* locus was verified by the absence of the WT band (*DDR48* diagnostic PCR; 2.11 kb), and the presence of the inserts (PCR *Prom_SIX5* - *Tgluc*; 0.58 kb).</caption>

<caption>**Supplemental Fig. S7.** Complementation of virulence of a Fol 007 ΔSix5 deletion strain with L. maculans SIX5 homologs.

Ten-day-old tomato seedlings were inoculated with water (mock), *Fol* 007 (WT), *Fol* 007 ΔSix5, or (cross-)complementation (CC) transformants (*SIX5*<sup>C</sup> T4, T6, T7; AvrLm10A<sup>CC</sup> T1, T2; AvrLm10A_like1<sup>CC</sup> T1, T6, T9; AvrLm10A_like2<sup>CC</sup> T1, T2). Fresh weight (FW) and disease index (DI) were assessed three weeks post inoculation; Fresh weight and disease index scores of the two individual biological replicates (replicate 1 N=15, replicate 2 N=20 plants per isolate/treatment) are shown. Continuous fresh weight data was tested for significant differences between the complementations, significant different groups were defined using TukeyHSD. Discrete disease index data was tested for significant differences between complementations using kruskal wallis, significant different groups were defined by pairwise wilcox test (non parametric rank sum test). The size of data points indicates tha number of similar disease index scores assigend. Grouping of the different complementations is indicated using letter codes above the graphs.</caption>
669 Supplemental tables

670

671 Supplemental Table 1. Quality of protein structure models predicted for the
672 AvrLm10A/AvrLm10B family.

673

674 Supplemental Table 2. Similarities between protein structures of the AvrLm10A/AvrLm10B
675 family.

676

677 Supplemental Table 3. Primer list.

678

679 Supplemental Table 4. Bayesian Information Criterion (BIC) values for cluster models.

680

681 Supplemental Table 5. Bootstrapping values for cluster model (EVI).

682

683

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