# TTargeting of the barley cell-surface receptor SRF3 by the Blumeria hordei effector AVRA13 overlaps with AVRA13 recognition by MLA and the induction of NLR-mediated cell death

Wei Shi, Merle Bilstein-Schloemer, Sara C Stolze, Matthieu P Platre, Maike Hofmann, Gunther Doehlemann, Hirofumi Nakagami, Isabel M L Saur

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To cite this version:

Wei Shi, Merle Bilstein-Schloemer, Sara C Stolze, Matthieu P Platre, Maike Hofmann, et al. TTargeting of the barley cell-surface receptor SRF3 by the Blumeria hordei effector AVRA13 overlaps with AVRA13 recognition by MLA and the induction of NLR-mediated cell death. 2026. hal-05457685

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Preprint submitted on 14 Jan 2026

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Targeting of the barley cell-surface receptor SRF3 by the *Blumeria hordei* effector *AVR*<sub>A13</sub> overlaps with *AVR*<sub>A13</sub> recognition by MLA and the induction of NLR-mediated cell death

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Wei Shi¹,§, Merle Bilstein-Schloemer¹,§, Sara C. Stolze², Matthieu P. Platre³, Maike Hofmann¹,

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Gunther Doehlemann¹,⁴, Hirofumi Nakagami²,⁵, Isabel M.L. Saur¹,⁴,§

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¹Institute for Plant Sciences, University of Cologne, 50674 Cologne, Germany.

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²Protein Mass Spectrometry, Max-Planck Institute for Plant Breeding Research, 50829

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Cologne, Germany.

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³IPSIM, Univ Montpellier, CNRS, INRAE, Institut Agro, 34060, Montpellier, France.

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⁴Cluster of Excellence on Plant Sciences (CEPLAS), Cologne, Germany.

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⁵Basic Immune System of Plants, Max-Planck Institute for Plant Breeding Research, 50829

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Cologne, Germany.

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⁶First authors

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⁷Correspondence: isabel.saur@uni-koeln.de

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20 Abstract

Pathogens secrete effector proteins to promote virulence. Despite their recognition by barley

*Mla* resistance genes, the structurally-related *Blumeria hordei* (*Bh*) AVR~A~ effectors are maintained in the *Bh* genome, suggesting virulence functions critical for fungal pathogenicity.

Using proximity-dependent protein labelling in transgenic barley, we detected distinct host protein interactomes for five AVR~As~ despite their structural homology and convergence on MLAs. We report the specific interaction of the highly conserved AVR~A13~ effector with the barley cell-surface receptor SRF3. AVR~A13~ disrupts HvSRF3-HvBAK1 interaction and alters HvSRF3 plasma membrane levels. AVR~a13~-expression desensitizes iron-induced restriction of *Bh* growth, suggesting that AVR~A13~ facilitates fungal proliferation by manipulating SRF3-mediated iron homeostasis. Our results suggest that MLAs have diversified to specifically detect the residues that underlie *Bh* effector neo-functionalization and intrinsic AVR~A~ virulence functions. Together, these findings identify SRF3 as molecular link between pathogen virulence, immune recognition, and iron homeostasis.

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41 **Introduction**

42 **Plant immune receptor-mediated non-self-recognition and pathogen virulence effectors**

43 To defend against pathogenic microbes, plants trigger immune responses upon detecting
44 conserved microbial patterns, such as fungal chitin or bacterial flagellin, via cell-surface
45 receptor-like kinases (RLKs), many of which use the co-receptor BAK1. This response is known
46 as pattern-triggered immunity (PTI)¹,². Plant-adapted microbial invaders secrete effectors to
47 suppress host immunity and acquire nutrients, which is the intrinsic effector activity³,⁴. Host
48 resistance to adapted pathogens is mediated by plant *Resistance (R)* genes, that frequently
49 encode intracellular nucleotide-binding oligomerization domain-like receptors (NLRs), which
50 detect pathogen effectors⁵. Effector-mediated activation of NLRs is frequently associated by
51 a localized cell death response⁶, which eliminates the host niche of biotrophic pathogens,
52 which feed of living host cells⁶. Pathogen effectors recognized by *R* genes are historically
53 known as ‘avirulence effectors’ (AVRs) and this activity can be considered the effector’s
54 avirulence function². Recognition of AVR s by direct interaction with an immune receptor is
55 typically associated with rapid evasion of NLR recognition by accumulation of effector gene
56 polymorphisms in pathogen populations⁶.

57 The obligate biotrophic powdery mildew fungi infect a wide range of plants including
58 economically relevant crops 7. The barley powdery mildew fungus *Blumeria hordei* (*Bh*)
59 harbors hundreds of candidate secreted effector protein (CSEP)-coding genes8,9. The large
60 number of CSEPs paired with the difficulties to genetically modify obligate biotrophs makes it
61 challenging to experimentally identify the effectors virulence function crucial for barley host
62 infection. This constitutes a major knowledge gap in molecularly understanding the
63 establishment of an obligate biotrophic lifestyle¹⁰. The diversified *Mildew locus a* encodes
sequence-related NLRs with recognition specificities towards multiple fungal effectors, 64 among them the *Bh* AVR<sub>A</sub> effectors<sup>11-13</sup>. For example, *Mla1* barley lines are resistant to all *Bh* 65 isolates that carry the *AVR*<sub>a1</sub> effector gene, but not to those isolates that have polymorphisms 66 in the *AVR*<sub>a1</sub> gene that resulted in escape from MLA1 recognition<sup>14,15</sup>. To date, seven AVR<sub>a</sub> 67 effectors have been isolated<sup>14-16</sup>. All belong to different subclasses of the expanded *Bh* RNase- 68 Like Proteins expressed in Haustoria (RALPHs) effector family<sup>14,16-18</sup>. Like most RALPH 69 members, AVR<sub>A</sub> effectors are pseudo-RNases lacking catalytic residues, which suggests that 70 they evolved from an ancient ribonuclease with neo-functionalization of virulence 71 functions<sup>8,19</sup>. The maintenance of AVR<sub>a</sub> gene variants in the *Bh* population, despite *Mla*- 72 imposed counterselection, suggests crucial virulence functions for these *Bh* CSEPs. Notably, 73 the escape from *Mla* recognition is predominantly mediated by nucleotide polymorphisms in 74 AVR<sub>a</sub> genes but not presence absence polymorphisms<sup>14-16</sup>, which further underlines this 75 assumption. In particular AVR<sub>a13</sub>, whose product is recognized by MLA13, is highly conserved 76 in the *Bh* population and variants with polymorphisms that could mediate the loss of 77 avirulence activity are exceptionally rare<sup>14,15,20</sup>. 78

We set out to isolate the host targets of *Bh* AVR<sub>A</sub> effectors and thereby investigate molecular 79 mechanisms employed by agriculturally important biotrophic cereal pathogens to establish 80 host infection. By employing transgenic barley lines stably expressing AVR<sub>a</sub> effectors we here 81 identify the host interactome of five AVR<sub>A</sub> effectors *via* a non-biased *in planta* biotin ligase- 82 based proximity-dependent protein labelling approach. Our data suggests that AVR<sub>A</sub> RNAse- 83 like effectors interact with divers sets of barley proteins. We identified the barley Strubbelig 84 Receptor Family 3 (HvSRF3) receptor-like kinase as specific interactor of AVR<sub>A13</sub>. AVR<sub>A13</sub> affects 85 HvSRF3 plasma membrane (PM) abundance and disrupts the association between HvSRF3 86 and the co-receptor HvBAK1. Importantly, we detect iron-related phenotypes upon AVR<sub>A13</sub> 87
expression in monocots and dicots, a phenotype also associated with SRF3 function. Finally, we show that the $AVR_{A13}$ region required for HvSRF3 interaction overlaps with the residues required for recognition by the MLA13 immune receptor. Together, our results suggest that MLAs have diversified to specifically detect the residues that underlie RALPH neo-functionalization and intrinsic $AVR_A$ virulence functions.

## 93 Results

### 94 Identification of $AVR_A$ interactors

To identify the $AVR_A$ interactome inside barley cells, we generated stable transgenic barley expressing each of the *Bh* $AVR_α$ effectors fused to the BirA biotin ligase and a 4myc tag ($AVR_A$-BirA). BirA exhibits high specificity in labelling $AVR_A$ association partners with biotin that can be isolated using immobilized streptavidin²⁰. We obtained barley transformants for $AVR_{a1}$-BirA, $AVR_{a7}$-2-BirA, $AVR_{a9}$-BirA, $AVR_{a10}$-BirA, $AVR_{a13}$-BirA, and the control line BirA. For each sample, barley leaves were infiltrated with 250 µM biotin and proteins were extracted under denaturing conditions prior to streptavidin-mediated isolation of biotinylated proteins and liquid chromatography tandem mass spectrometry (LC-MS/MS)-based identification of isolated proteins (Supplementary Fig. 1a)²¹. Transgene expression and biotinylation of barley proteins by BirA and $AVR_A$-BirA was confirmed by anti-myc and Strep-HRP western blot after total protein extraction (Supplementary Fig. 1b-1h). This experimental setup was performed independently for each AVR effector and control using two independent transgenic lines. Thereby we identified 61, 45, 65, 77 and 118 and proteins as associators of $AVR_{A1}$, $AVR_{A7}$-2, $AVR_{A9}$, $AVR_{A10}$, and $AVR_{A13}$, respectively (Fig. 1a, Supplementary Fig. 2). We did not observe significant overlaps among interactomes of the different $AVR_A$ effector datasets, suggesting that the isolated *Bh* effectors target different sets of proteins in the barley host (Fig. 1b).
111 **AVR<sub>A13</sub> associates with barley Strubbelig Receptor Family 3 (SRF3)**

112 *AVR*<sub>a13</sub> is the most highly conserved *AVR*<sub>a</sub> in the global *Bh* population based on the
113 diversifications associated with MLA virulence phenotypes<sup>14,15</sup>, which suggests a crucial
114 virulence function of this effector. Accordingly, we consistently detected a significantly higher
115 *Bh* infection rate on barley lines expressing *AVR*<sub>a13</sub> when compared to control plants and
116 respective negative sister lines (Fig. 1c and 1d). We thus set out to further investigate the
117 *AVR*<sub>A13</sub> function by mining *AVR*<sub>A13</sub> associating proteins. We detected variable enrichment
118 levels of putative *AVR*<sub>A13</sub> interactors between proximity labelling experiments that use
119 independent *AVR*<sub>a13</sub> transgenic lines (Supplementary Data 1). However, across all datasets
120 obtained from both independent transgenic lines, one protein that identified as barley
121 STRUBBELIG RECEPTOR FAMILY 3-like (HvSRF3) was highly enriched in the *AVR*<sub>A13</sub> samples
122 when compared to both, BirA and wild-type control samples (Fig. 1e, Supplementary Data 1).
123 SRF3 is a PM-localized leucine-rich repeat receptor-like kinase (LRR-RLK) with a unique N-
124 terminal STRUBBELIG (SUB) domain of unknown function<sup>22</sup>. To get first insight if HvSRF3 might
125 play a role during *Bh* infection of barley, we determined HvSRF3 subcellular localization upon
126 *Bh* attack upon expression of annotated *HvSRF3* (LOC123401201) in single barley epidermal
127 cells using biolistic delivery. Transformed cells were visualized by HvSRF3-mYFP expression in
128 the absence of *Bh*. However, it appeared difficult to localize HvSRF3 signals in *Bh* infected
129 cells. By detecting infected and non-infected *HvSRF3*-expressing cells within close proximity,
130 we found that *Bh*-infected cells appeared to display reduced HvSRF3 signal when compared
131 to neighboring non-infected cells (Fig. 1f). Thus, HvSRF3 PM abundance may be altered during
132 *Bh* infection.

133 **AVR<sub>A13</sub> binds the cytoplasmic domain of barley Strubbelig Receptor Family 3**
Four barley LRR-RLKs are annotated as HvSRF3-like²³, and we named these HvSRF3-Like1 (HvSRF3-L1, LOC123409827), HvSRF3-Like2 (HvSRF3-L2, LOC123401201), HvSRF3-Like3 (HvSRF3-L3, LOC123425675) and HvSRF3-Like4 (HvSRF3-L4, LOC123403104) (Fig. 2, Supplementary Fig. 3, Fig. Supplementary 4). By searching available barley databases (NCBI, EnsemblPlants and Uniprot), we identified a total of 12 SRF-like genes. Sequence comparison revealed that these are orthologous to A. thaliana SRF2, SRF4, SRF5, SRF6, SRF7 and SRF8 and that the previously annotated HvSRF3 proteins are homologous to A. thaliana SRF3, although we also detected homology between HvSRF3-L4 and AtSRF9 (AtSUB, Fig. 2a and 2b, Supplementary Fig. 3)²².

In the AVR<sub>A13</sub> proximity labelling datasets, we only identified peptides specific to HvSRF3-L1 and HvSRF3-L2 (Supplementary Data 1). To determine if HvSRF3-L1 and HvSRF3-L2 indeed interact with AVR<sub>A13</sub>, we tested their association with AVR<sub>A13</sub> and its naturally occurring virulent variant AVR<sub>A13</sub>-V2 by luciferase complementation (split-luciferase assay) upon co-expression of respective constructs in Nicotiana benthamiana leaves. We detected luciferase activity upon co-expression of *AVR*<sub>a13</sub> and the virulent variant *AVR*<sub>a13</sub>-V2<sup>14,15,20</sup> with both HvSRF3-L1 and HvSRF3-L2 (Fig. 2c). Similarly, immunoprecipitation of AVR<sub>A13</sub>-mYFP and AVR<sub>A13</sub>-V2-mYFP but not GFP co-isolated HvSRF3-L1-6HA and HvSRF3-L2-6HA (Fig. 2d). Both HvSRF3 proteins appear n-terminally fragmented and HvSRF3-L1-6HA protein is less stable than HvSRF3-L2 upon *in planta* expression (Fig. 2d; Supplementary Fig. 5a).

Because AVR<sub>A13</sub> is recognized by MLA13 inside barley cells<sup>14,15</sup>, it is plausible to assume that AVR<sub>A13</sub> interacts with the cytoplasmic domain (CD) of HvSRF3. To test this, we performed east two-hybrid (Y2H) assays with AVR<sub>A13</sub> and the cytoplasmic domains of HvSRF3-L1 (HvSRF3-L1<sup>CD</sup>) and HvSRF3-L2 (HvSRF3-L2<sup>CD</sup>). We used MLA13 as a control and as expected, both AVR<sub>A13</sub>
and $AVR_{A13}$-V2, but not GFP interacted with MLA13 (Fig. 2e)¹⁴,²⁰. We also detected interaction of $AVR_{A13}$ and $AVR_{A13}$-V2 with HvSRF3-L1<sup>CD</sup> and HvSRF3-L2<sup>CD</sup> (Fig. 2e, Supplementary Fig. 5b). None of the HvSRF3-Like proteins were biotinylated by other *Bh* $AVR_A$ effectors in the proximity labelling experiment (Fig. 1b, Supplementary Data 1). We conclude that HvSRF3 is a specific target of $AVR_{A13}$.

**The specificity of the interaction between $AVR_{A13}$ and HvSRF3 is mediated by the HvSRF3 kinase domain**

We neither detected HvSRF3-L3, nor HvSRF3-L4 in the $AVR_{A13}$ proximity labelling samples (Supplementary Data 1). To determine the general capacity of $AVR_{A13}$ to bind HvSRF3-L3 and HvSRF3-L4, we performed Y2H assays with *HvSRF3-L3*<sup>CD</sup> and *HvSRF3-L4*<sup>CD</sup> and found that HvSRF3-L3<sup>CD</sup> but not HvSRF3-L4<sup>CD</sup> could interact with $AVR_{A13}$ and $AVR_{A13}$-V2 (Fig. 3a, Supplementary Fig. 5c). The cytoplasmic regions of HvSRF3-like proteins consist of the C-terminal kinase domains (KDs) and the juxtamembrane domains (JMs) that encompass a promiscuous proline-rich-region (PRR) (Fig. 2, Supplementary Fig. 3, Supplementary Fig. 4)²². Sequence analysis demonstrates high variability within the JM domain across the HvSRF3-Like proteins with HvSRF3-L4<sup>JM</sup> being shorter than the JMs of the other three HvSRF3-Like proteins (Supplementary Fig. 4). To understand if the differences in the JM domain account for the lack of interaction between $AVR_{A13}$ and HvSRF3-L4, we tested if $AVR_{A13}$ can bind the HvSRF3-L1<sup>KD</sup> and HvSRF3-L2<sup>KD</sup>, which represent the CDs without respective JMs. Neither $AVR_{A13}$ nor $AVR_{A13}$-V2 showed interaction with HvSRF3-L1<sup>KD</sup> or L2<sup>KD</sup> (Fig. 3b, Supplementary Fig. 5d). To further clarify the roles of the individual HvSRF3 domains in mediating the $AVR_{A13}$ association, we generated hybrid constructs exchanging the JM domains of HvSRF3-L1 and HvSRF3-L2 with the HvSRF3-L4<sup>JM</sup> and *vice versa* (Fig. 3c). Only the hybrids encoding the HvSRF3-L1<sup>KD</sup> or
180 HvSRF3-L2KD interacted with AVR<sub>A13</sub> (Fig. 3d and 3e, Supplementary Fig. 5e). We conclude that
181 AVR<sub>A13</sub> requires the JM of HvSRF3 for interaction in yeast, but that specificity of the interaction
182 between AVR<sub>A13</sub> with HvSRF3-L1 and HvSRF3-L2 is mediated by the respective HvSRF3KD.
183 Interestingly, kinase activity could not be detected for SRF3 and other SRFs from A. thaliana
184 <sup>24-27</sup>. We therefore evaluated the general propensity of the HvSRF3KDs for putative
185 phosphorylation capacity by assessing the conservation of key kinase residues. We visualized
186 the KD structures (AlphaFold3) of HvSRF3 and the kinase active receptor HvBAK1
187 (BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1) (Fig 3f-h). The analyses
188 demonstrate that in addition to the protein polymorphisms within the conserved catalytic
189 loop of AtSRF3 when compared to the kinase active BAK1<sup>KD</sup>s, HvSRF3KDs lack both, the GxGxxG
190 motif of the P-loop and the DFG motif. Correspondingly, neither Mg<sup>2+</sup> nor ATP is not modelled
191 into the HvSRF3 binding pockets (Fig. 3f to 3h, Supplementary Fig. 5f)<sup>28</sup>. We conclude that,
192 similar to AtSRF3 and AtSRF9, the KDs of HvSRF3s are most likely pseudokinases. SRF3 and
193 other SRFs may thus act as molecular scaffolds for other RLKs or rely on the kinase activity of
194 associating co-receptors with active kinases and we speculate that either of these putative
195 HvSRF3 functions is manipulated by AVR<sub>A13</sub>.

196 **AVR<sub>A13</sub> affects HvSRF3-HvBAK1 interaction and HvSRF3 arrangement at the plasma**
197 **membrane**

198 Cell surface-localized LRR-RLKs frequently rely on the co-receptor BAK1/SERK3 (SOMATIC
199 EMBRYOGENESIS RECEPTOR KINASE3) or other SERK family members to transduce signals
200 across the PM<sup>29,30</sup>. Because multiple SRF members have been reported to associate with
201 BAK1<sup>31-33</sup>, we hypothesized that HvSRF3 might rely on HvBAK1 kinase activity to transduce
202 signals across the PM. We therefore cloned HvBAK1 (EF216861.1)<sup>34</sup> and determined if HvSRF3
and HvBAK1 associate upon co-expression. For this, we primarily focused on HvSRF3-L2 (hereafter referred to as HvSRF3), because the HvSRF3-L1 protein is poorly detectable upon *in planta* expression (Fig. 2d; Supplementary Fig. 5a). Y2H analysis involving HvBAK1<sup>CD</sup> and HvSRF3-L2<sup>CD</sup> (Fig. 4a, Supplementary Fig. 6a) and split-luciferase assays of full length HvBAK1 and HvSRF3-L2 in *N. benthamiana* leaves demonstrated interaction of HvSRF3 and HvBAK1 (Fig. 4b). In agreement, we detected fluorescence signals via YFP complementation by co-expression of HvBAK1 fused to the C-terminal domain of YFP (HvBAK1-YFP<sup>C</sup>) and HvSRF3 fused to the N-terminal domain of YFP (HvSRF3-YFP<sup>N</sup>, Fig. 4c). Several pathogen effectors manipulate receptor signaling outputs by modulating association with the BAK1 co-receptor<sup>35</sup>. We therefore investigated if AVR<sub>A13</sub> can manipulate association of HvSRF3 with HvBAK1. In split-luciferase assays, signals mediated by HvBAK1-cLUC and HvSRF3-nLUC were abrogated in the presence of AVR<sub>A13</sub>-mYFP and AVR<sub>A13</sub>-V2-mYFP but not GFP (Fig 4d and 4e, Supplementary Fig. 6b). We also tested the specificity of this effect by employing CSEP0371, which is the *Bh* effector most closely related to AVR<sub>A13</sub> (also known as CSEP0372)<sup>8</sup> and found that CSEP0371-mYFP does not affect luciferase signal of HvBAK1-cLUC and HvSRF3-nLUC (Fig 4d and 4e, Supplementary Fig. 6b). Similar results were obtained in a YFP complementation assay, where the YFP signal that was detectable upon co-expression of HvBAK1-YFP<sup>C</sup> and HvSRF3-YFP<sup>N</sup> was largely diminished in the presence of AVR<sub>A13</sub>-mCherry but not mCherry alone (Fig. 4f and 4g, Supplementary Fig. 6c and 6d). We detected fluorescent signal at the PM but also in the cytosol and also concentration of the receptor complex in specific regions at the PM in the presence of AVR<sub>A13</sub> (Fig. 4f and 4g, Supplementary Fig. 6e). Thus, we tested if AVR<sub>A13</sub> is able to mediate changes to HvSRF3 PM organization, a process associated with AtSRF3 function in *A. thaliana* roots<sup>36</sup>. We co-expressed HvSRF3-mCherry with GFP or either effector and found that in comparison to GFP, HvSRF3 PM signals but not total HvSRF3 protein
levels are reduced in the presence of AVR<sub>A13</sub> and AVR<sub>A13</sub>-V2 but not CSEP0371 (Fig. 4h to 4j,
Supplementary Fig. 6f). Effector signals were also detected in the nucleus (Fig. 4h,
Supplementary Fig. 6c and 6e). We cannot clarify if AVR<sub>A13</sub>-medited HvSRF3 PM organization
is the cause or a consequence of abrogated HvSRF3-HvBAK1 interaction. However, both
observations point towards negative regulation of HvSRF3 by AVR<sub>A13</sub>.

232 **AVR<sub>A13</sub>-mediated modulation of SRF3 is conserved between monocots and dicots**

Despite high levels of conservation across land plants³⁷, the molecular functions of SRF
members are not fully understood. Recently, rice SRF3 has been implicated to regulate chitin-
induced PTI responses³³. In protoplasts derived from rice lines overexpressing OsSRF3, chitin-
induced production of reactive oxygen species (ROS) was elevated, and this was affected by
the effector UgsL from the fungal pathogen *Ustilaginoidea virens*³³. To determine if AVR<sub>a13</sub>
modulates typical PTI output to mediate pathogen virulence, we assayed chitin-mediated ROS
production using the stable *AVR*<sub>a13</sub>-expressing barley lines with increased susceptibility to *Bh*.
We did not detect significant differences in chitin-mediated ROS production between control
or *AVR*<sub>a13</sub>-expressing barley lines (Supplementary Fig. 7).

In *A. thaliana*, AtSRF3 regulates iron homeostasis and nutritional immunity<sup>25,36</sup>. AtSRF3-
mediated sensing of low iron signals is also associated with reduced AtSRF3 levels at the PM,
and *srf3* lines are insensitive to low iron phenotypes such as low iron-mediated root growth
inhibition<sup>36</sup>. Low iron levels have also been reported within barley cells that are attacked by
powdery mildews<sup>38</sup>. We therefore decided to genetically determine if *AVR*<sub>a13</sub>-expression can
affect SRF3 phenotypes at low iron in *A. thaliana*. We found that *AVR*<sub>A13</sub> can indeed bind
AtSRF3 using split-luciferase, co-immunoprecipitation, and Y2H assays (Fig. 5 a, b and c,
Supplementary Fig. 8a). Thus, we generated homozygous *AVR*<sub>a13</sub>-expressing *A. thaliana* Col-0
lines (Fig. 5d, Supplementary Fig. 8b) and determined low iron-mediated root growth response³⁶. At physiological iron levels, *AVR*<sub>a13</sub> expressing *A. thaliana* seedlings did not show root growth inhibition (Fig. 5e). However, the low-iron induced root growth inhibition response was impaired in *AVR*<sub>a13</sub> expressing lines and this mimics *srf3* mutant phenotypes (Fig. 5f)³⁶. Together, the data suggests that *AVR*<sub>A13</sub> can impair SRF3 function in regulating iron deficiency responses in dicots.

Iron uptake and transport strategies, as well as iron deficiency responses differ greatly between dicots such as *A. thaliana* and monocots such as barley and compared with roots, our understanding of iron transport and homeostasis in leaf tissues is limited³⁹⁻⁴¹. Yet, powdery mildew attack was shown to cause local intracellular iron depletion and extracellular increase of ferric iron, which is associated with the production of reactive oxygen species such as H₂O₂ and nitric oxide³⁸. However, it is unclear how these fungal-induced alterations to host iron homeostasis affect the outcome of such fungal penetration attempts and powdery mildew disease development. To get first insight into this, we infected barley grown at differential iron levels with *Bh*. Plants grown under low iron conditions (10 µM Fe-EDTA, which represents 10 x less iron than the control sample) were not significantly impacted in *Bh* susceptibility, whereas barley grown at high levels of iron (250 µM Fe-EDTA, which represents 2.5 x more iron than the control sample) showed reduced *Bh* infections rates (Supplementary Fig. 8c). To mimic host induced iron accumulation at the actual site of penetration and counteract intracellular iron depletion without disturbing the whole plant's iron status, we applied chelated iron (FeEDDHA) to barley leaves prior to *Bh* infection. We found that this also leads to reduced *Bh* disease symptoms (Fig. 5g and 5h). Importantly, in contrast to controls, barley lines expressing *AVR*<sub>a13</sub> were insensitive to the decreased fungal infection upon FeEDDHA application (Fig. 5i and 5j). From this, we conclude that high levels of iron
274 negatively influence *Bh* proliferation and that AVR<sub>A13</sub> desensitizes fungal susceptibility to high iron levels. We hypothesize that, for promoting virulence, AVR<sub>A13</sub> targets SRF3 to manipulate iron sensing in *Bh* infected plant cells or iron-associated processes such as the oxidative burst at the site of fungal infection.
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278 **AVR<sub>A13</sub> residues required for SRF3 interaction are recognized by MLA13**

279 All AVR<sub>A</sub> effectors analyzed here belong to the *Bh* RALPH effector family for which it was 280 postulated that multiple members can target the same host processes to promote 281 virulence<sup>18,42</sup>. AVR<sub>A13</sub> (also known as CSEP0372) has three family members in *Bh*, namely 282 CSEP0371, CSEP0373 and CSEP0374 (Fig. 6a and 6b)<sup>8</sup>. CSEP0371 and CSEP0374 are expressed 283 during *Bh* infection of barley, but CSEP0373 is not<sup>9</sup>. We already demonstrated that unlike 284 AVR<sub>A13</sub>, CSEP0371 does not affect HvSRF3 phenotypes (Fig. 4). To directly determine CSEP 285 family-wide conservation of *Bh* effector targets, we tested interaction of CSEP0371 and 286 CSEP0374 with the HvSRF3-L1<sup>CD</sup> which showed the strongest interaction with AVR<sub>A13</sub> in Y2H 287 assays (Fig. 2e). We failed to detect interaction of CSEP0371 and CSEP0374 with HvSRF3-L1<sup>CD</sup> 288 in Y2H (Fig. 6c, Supplementary Fig. 9a). We then made use of this to identify the HvSRF3- 289 binding residues in AVR<sub>A13</sub>. For this, we generated eight chimeric AVR<sub>A13</sub> / CSEP0371 290 constructs (chimera 1-8, Fig. 6d). Interaction of chimera 6 and chimera 8 but not chimera 4 291 with the HvSRF3<sup>CD</sup> in the Y2H assay suggests that a central loop region of the AVR<sub>A13</sub> protein 292 mediates HvSRF3 interaction (Fig. 6e, Supplementary Fig. 9b). Interestingly, this region 293 contains seven of 11 residues that were previously identified as MLA13 contact residues<sup>43</sup>. 294
Based on these observations, we hypothesized that these MLA13 contact residues may also 295 play a role in the interaction of AVR<sub>A13</sub> with HvSRF3. We therefore exchanged corresponding 296 AVR<sub>A13</sub> residues to CSEP0371 residues (chimera 10) and *vice versa* (chimera 9). Additionally,
we generated chimeric constructs to more specifically define the region of AVR<sub>A13</sub> that
mediates HvSRF3 interaction (chimera 11, 12 and 13). As determined by the use of chimera
9, the introduction of the AVR<sub>A13</sub>-MLA13 contact residues into CSEP0371 alone was
insufficient for transferring the ability of AVR<sub>A13</sub> to bind HvSRF3. On the other hand, the
elimination of the MLA13 contact residues in AVR<sub>A13</sub> in chimera 10 disrupted effector binding
to HvSRF3 (Fig. 6e, Supplementary Fig. 9b), implying that the MLA13 contact residues of
AVR<sub>A13</sub> play a crucial role for association with HvSRF3. Chimera 10 to chimera 13 narrowed
down the functional region for HvSRF3 interaction to AVR<sub>A13</sub> amino acid (aa) E44 to aa N70,
which is largely composed of a central loop region (Fig. 6h and 6i). Further exchanges of
individual AVR<sub>A13</sub> amino acids to corresponding CSEP0371 residues in this region (chimera 14
and 15, Fig. 6d) abrogated interaction with HvSRF3<sup>CD</sup> (Fig. 6e). Interestingly, of all 11 MLA13
contact residues in AVR<sub>A13</sub>, only five residues abrogate MLA13 activation upon exchange to
alanine <sup>43</sup>. All of these five 'AVR<sub>A13</sub> avirulence residues' locate within the AVR<sub>A13</sub> central loop
region required for interaction of AVR<sub>A13</sub> with HvSRF3 ('AVR<sub>A13</sub> virulence residues'). We
therefore hypothesized that the AVR<sub>A13</sub> avirulence residues overlap with AVR<sub>A13</sub> virulence
residues. To test this, we determined the ability of the here generated chimeras to activate
MLA13. For this, we co-expressed the constructs in *N. benthamiana* leaves for macroscopic
evaluation of MLA13-mediated cell death. Additionally, we expressed the constructs in barley
protoplasts to quantitatively assess MLA13-mediated cell death by determining reduction of
luciferase reporter activity as proxy for cell viability. As expected, CSEP0371 did not activate
MLA13 cell death and only the chimeras that can bind HvSRF3 activated MLA13 to the same
extend as native AVR<sub>A13</sub> (Fig. 6f and 6g, Supplementary Fig. 9c). Notably, with respect to
chimera 12 and chimera 14, the exchange of the amino acids E44 and H45 of AVR<sub>A13</sub> to R44
and D45 of CSEP0371 significantly impaired activation of MLA13 and abrogated association
with HvSRF3 underlining a central role of these residues in AVR<sub>A13</sub> avirulence and presumed virulence function on HvSRF3 (Fig. 6f and 6g). We conclude that the central region encoded by AVR<sub>A13</sub> aa E44 to aa N70 (Fig. 6h and 6i) is required for activation of MLA13 and interaction with the host target HvSRF3 and therefore for an SRF3-mediated virulence function of the effector. It is thus plausible to assume that MLA13 has specifically evolved to detect the region of AVR<sub>A13</sub> required for inhibition of HvSRF3 function.

## Discussion

We report the interactomes of five RNase-like *Bh* AVR<sub>A</sub> effectors in the barley host. Despite their structural homology, individual AVR<sub>A</sub> effectors engage with distinct sets of host proteins (Fig. 1, Supplementary Data 1). These findings experimentally support the hypothesised neo-functionalization of *Bh* RALPH effectors and align with previous studies that independently identified variable host targets for RALPH family members<sup>10</sup>. By deeper investigation of the most conserved *Bh* avirulence effector AVR<sub>A13</sub>, we demonstrate virulence activity and association with the CD of the barley RLK SRF3 for which the KD was shown to encode binding specificity (Fig. 2 and Fig. 3). This activity cannot be performed by AVR<sub>AS</sub> that are recognized by other *Mla* alleles (Fig. 1), suggesting variable virulence functions for individual AVR<sub>A</sub> effectors. Collectively, our results suggest that pathogen effectors that have originated from the same species and have substantially contributed to the diversification of host NLR receptors, do not necessarily converge on the same host targets for virulence.

Similar to the *Bh* AVR<sub>A</sub> RALPH effectors, the *Magnaporthe oryzae* AvrPik effectors recognized by allelic variants of Pik1 are structural homologs<sup>44-46</sup>. Pik1 detects allelic AvrPik variants through the integrated Heavy Metal-Associated (HMA) domain and the allelic AvrPik
effectors exert virulence activity by binding the rice heavy metal-associated isoprenylated proteins (HIPPs) OsHIPP19 and OsHIPP20<sup>46,47</sup>. This interaction is conserved across all tested AvrPik variants, suggesting that Pik1 evolved to recognize AvrPik virulence activity by mimicking HIPP19 and HIPP20 HMA domains <sup>47</sup>. In turn, the divergent *M. oryzae* effectors AVR-Pia, AVR1-CO39 and Pwl2 cannot bind HIPP19 and HIPP20 and PWL2 instead associates with OsHIPP43<sup>47,48</sup>. This prompts us to speculate that *Bh* AVR<sub>A</sub> and potentially other RALPH effectors may likewise converge on specific domains within distinct barley proteins to promote *Bh* virulence. We show that the specificity of AVR<sub>A13</sub> association with HvSRF3 is determined by the KD of HvSRF3, suggesting that KD-containing proteins that participate in diverse physiological processes, may serve as convergent targets for AVR<sub>A</sub> effectors. Even within the AVR<sub>A13</sub> effector family, HvSRF3 associates only with AVR<sub>A13</sub>, despite CSEP0371 and CSEP0374 being the most closely related *Bh* effector proteins in terms of sequence and structure (Fig. 6)<sup>8</sup>. This observation is significant because prior structural analyses of AVR<sub>A</sub> effectors led to the hypothesis that members of the same RALPH subclass share conserved virulence activities<sup>18</sup>. It will be interesting to determine whether CSEP0371 and CSEP0374 interact with other HvSRFs or target distinct KD-containing proteins.

The association specificity of AVR<sub>A13</sub> with HvSRF3 is determined by the HvSRF3 KDs, which appear to be pseudokinases (Fig. 3). Similarly, no kinase activity has been detected for AtSRF3 *in vitro*<sup>36</sup> and the SRF3 member AtSUB/AtSRF9 appears to carry a catalytically inactive kinase domain<sup>24,26,27</sup>. Despite this, SRF3 and SUB KDs are required for SRF-specific phenotypes<sup>24,26,27,36</sup>. Based on the interaction of HvSRF3 and HvBAK1, we speculate that HvSRF3 might rely on HvBAK1 for signaling. This is consistent with multiple independent studies involving different SRF family members<sup>31-33</sup>. The AVR<sub>A13</sub>-mediated inhibition of the
366 HvSRF3-HvBAK1 association suggests that this interaction is indeed of biological relevance.

367 We thus propose that HvSRF3 requires HvBAK1 for downstream signaling, or alternatively,
368 regulates HvBAK1 function and that AVR<sub>A13</sub> interferes with this process.

369 The HvBAK1-HvSRF3 interaction targeted by a conserved mildew effector is notable, as
370 tandem kinase genes have recently been shown to mediate resistance to biotrophic fungi in
371 barley and wheat<sup>49</sup>. While not confirmed for all cases, tandem kinase effector recognition
372 seems to induce conformational changes within paired KDs<sup>49-51</sup>. This is relevant, as tandem
373 kinases typically pair an active kinase with a pseudokinase<sup>51-57</sup>, A configuration conceptually
374 similar to our observations, where AVR<sub>A13</sub> binding to the HvSRF3 pseudokinase domain
375 influences its association with the active HvBAK1 kinase. Tandem-kinase-recognized effectors
376 may enhance fungal virulence by targeting immune RLK complexes with active and inactive
377 kinases (e.g., BAK1/SERKs and SRFs).

378 AVR<sub>a13</sub>-expressing A. thaliana lines mimic srf3 mutants under low iron conditions (Fig. 5),
379 suggesting the conservation of the AVR<sub>A13</sub> function on SRF3 between dicots and monocots
380 and underlines negative regulation of SRF3 by AVR<sub>A13</sub><sup>36</sup>. A. thaliana srf3 mutants exhibit
381 increased iron levels under sufficient iron conditions. In turn, AtSRF3 is involved in the
382 upregulation of low iron response genes under iron deficient conditions<sup>36</sup>. As such, SRF3
383 responds to differential iron levels and regulates iron homeostasis. Importantly, SRF3-
384 mediated upregulation of low iron response genes is impaired during PTI responses<sup>36</sup>.

385 The regulation of iron homeostasis upon microbial attack is crucial for all hosts. Reducing the
386 iron content in the sites of infection to deprive the pathogens of iron (nutritional immunity)
387 and also the over-accumulation of iron at the infection sites to enhance the production of
388 physically damaging ROS are effective host defense strategies<sup>58</sup>. Both, intracellular ferrous
389 iron depletion and accumulation of extracellular ferric iron early during powdery mildew
390 attack has been reported³⁸. Here we found that reduced host iron availability has little effect
391 on *Bh* proliferation, suggesting that withholding iron is not an effective *Bh* defense strategy.

392 In turn, high levels of iron negatively regulate *Bh* infection (Fig. 5, Supplementary Fig. 8c) and
393 *AVR*<sub>A13</sub>-expressing barley lines are insensitive to this. We thereby link *AVR*<sub>A13</sub> function to
394 fungal iron sensitivity. Based on this, we hypothesize that SRF3 conveys information about
395 changes to iron levels in *Bh* infected cells and that this process i) might involve HvBAK1 and
396 ii) is inhibited by *AVR*<sub>A13</sub> to obstruct the resupply of immune active iron to the site of infection.

397 Notably, it is possible that *AVR*<sub>A13</sub> targets SRF3 to indirectly manipulate iron-associated
398 processes such as ROS production upon successful fungal penetration of host cells.

399 *AVR*<sub>A13</sub> binds HvSRF3 *via* a loop that also triggers MLA13-mediated cell death. Chimera and
400 cryo-EM data show residues outside this loop are dispensable for MLA13 activation (Fig. 6)⁴³,
401 suggesting MLA13 specifically evolved to recognize the SRF3-interaction loop of *AVR*<sub>A13</sub>, while
402 the MLA13 winged-helix domain enables general RNase-like effector binding. *AVR*<sub>A13</sub>'s
403 overlapping avirulence-virulence function likely prevents *Mla13*-evading variants, accounting
404 for its conservation in *Bh* isolates across decades¹⁵,¹⁴. In turn, virulent *AVR*<sub>A13</sub>-V2 does not
405 activate MLA13-mediated cell death but carries the shared HvSRF3-binding and MLA13-activity
406 loop region. We have previously demonstrated that *AVR*<sub>A13</sub>-V2 maintained the residues for
407 MLA13 interaction but blocks MLA13-mediated cell death and have thereby described a rare
408 pathogen-derived mechanism for escaping NLR-mediated resistance ²⁰. Our here presented
409 data can explain that this complex mechanism allows the escape from NLR-mediated
410 resistance whilst maintaining overlapping virulence propensities, which here appears
411 superior to the escape from NLR-binding¹⁴.
The overlap between HvSRF3-binding residues of AVR<sub>A13</sub> and those required for MLA13 activation highlights SRF3's central role in plant immunity. This is also supported by independent lines of evidence: (i) the involvement of SRF3 in autoimmunity mediated by the DM2h NLR in *A. thaliana* ecotype *Ler*<sup>59,60</sup>, suggesting that AtSRF3 acts as a decoy for an unknown effector<sup>61</sup>; (ii) the regulation of nutritional immunity by AtSRF3 in *A. thaliana* roots; and (iii) the targeting of rice SRF3 by an effector from a smut fungus<sup>33</sup>. Taken together, these findings highlight SRF3 as a conserved and pivotal regulator of plant-microbe interactions, with roles that extend well beyond *Bh* infection in barley. The ability of AVR<sub>A13</sub> to bind AtSRF3 will provide us with a genetically tractable system to further dissect SRF3 function during pathogen attack.

## Methods

### Plant material and growth

Barley was grown at 19°C, 70% relative humidity, and under a 16 h photoperiod. *Nicotiana benthamina* was grown as described<sup>62</sup>. *Arabidopsis thaliana* plants were grown at 21 °C, under an eight-hour photoperiod for experimental approaches or a 16-hour photoperiod for seed propagation.

### *Blumeria hordei* maintenance and infections

Maintenance of *Blumeria hordei* isolate K1 was carried out as described previously<sup>15</sup>. For fungal propagation and infection assays, one-week-old barley plants were transferred to an incubator with *Bh* isolate K1 infected barley cultivar Golden Promise incubator and thereby inoculated. For iron treatment, plants were sprayed with water containing 0.1% (v/v) Tween 20 (mock) or 0.5 g/L FeEDDHA solution containing 0.1% (v/v) Tween 20 and thereafter
434 transferred to the *Bh* incubator. For quantification of infection, the adaxial and abaxial surfaces of the first leaf were photographed, and *Bh* colonies were quantified five days post-inoculation.

437 **Blumeria hordei** infection and quantification on *in vitro* grown barley

438 For *in vitro* growth, barley seeds were surfaces sterilized in 6% sodium hypochlorite shaking
439 for one hour, washed five times with sterile Milli-Q water and germinated on wet sterile
440 Whatman filter paper. Four-day-old germinated seedlings were transferred into Weck glasses
441 containing sterile ½ MS medium with either 10 µM, 100 µM or 250 µM FeNaEDTA. Except for
442 FeNaEDTA, MS medium was prepared according to the recipe of Duchefa (M0255). Plants
443 were grown for another three days prior to infection with *Blumeria hordei* (*Bh*) isolate K1. At
444 seven days post infection, leaf tissue from three independent plants was pooled and frozen
445 in liquid nitrogen. Tissue was ground to a fine powder using a Retsch Beat Beater (2 × 1 min
446 at 30 Hz) and total DNA was extracted according to Edwards & Thompson 1991⁶³. Fungal
447 biomass was quantified by qPCR using the GoTaq® qPCR kit containing 2X BRYT Green® dye
448 master mixes, on the Bio-Rad CFX Connect Real-Time PCR System. *Bh* GAPDH primers
449 (5'GGAGCCGAGTACATAGTAGAGT'3, 5'GGAGGGTGCCGAAATGATAAC'3; GenBank:
450 CAUH01004767) and barley GAPDH primers (5'TTTGGTATTATTGAGGGTCTGATGA'3,
451 5'TGCTGCTGGGGATGATGTT'3; GenBank: X60343.1) were used. Data were visualized and
452 processed in Bio-Rad CFX Manager. Fold changes were calculated using the delta-Ct method
453 after normalization of the *Bh* GAPDH Ct values to the Ct mean of the barley GAPDH
454 housekeeping gene.

455 Generation of expression constructs
456 SRF3 variants, CSEP00371, CSEP00374, and the AVR<sub>a13</sub>-CSEP00371 chimeras were either
457 amplified from cDNAs of *A. thaliana* Col-0 or barley cv. Golden Promise using Q5 High-Fidelity
458 DNA Polymerase (NEB) or synthesized from GeneArt (Thermo Scientific) as pDONR221 entry
459 clones or with overhangs for restriction/ligation into Golden Gate Level 0 vectors<sup>64</sup>. For stable
460 transformation of barley, synthesized *BirA-4myc* and *AVR<sub>α</sub>-BirA-4myc*<sup>20</sup> constructs were
461 transferred from donor vectors into the expression vector pIPKb002<sup>65</sup>. The generation of
462 *pAtUBI10:FLAG-AVR<sub>α</sub>* constructs for expression of *AVR<sub>a13</sub>* in *A. thaliana* was described
463 previously<sup>15</sup>. For determination of HvSRF3 localization in barley, HvSRF3 constructs were
464 cloned together with *pZmUbi* promotor (derived from the pIPKb002 vector)<sup>65</sup> and a C-
465 terminal mYFP tag derived from the pXCSG-GW-mYFP vector<sup>66</sup> into pICH47732<sup>64</sup> by restriction
466 enzyme digest/ligation. For Y2H interaction studies, respective genes were either described
467 previously<sup>14</sup> or were transferred from gateway entry or donor vectors into the expression
468 vectors pLexA-GW, or pB42AD-GW<sup>14</sup> using LR Clonase II (Thermo Scientific). For split-
469 luciferase assays, respective genes were cloned into expression vector pCAMBIR1300-nLUC
470 or pCAMBIR1300-cLUC by NEBuilder HiFi DNA Assembly as described<sup>21</sup>. For CoIP assays, SRF3
471 constructs, the cauliflower mosaic virus 35S promoter sequence and a 6HA or GFP tag
472 sequences were cloned into expression vector pICH47732<sup>64</sup> using restriction enzyme
473 digestion/ligation or NEBuilder HiFi DNA Assembly. AVR<sub>A13</sub>-mYFP constructs were described
474 previously<sup>14</sup>. For BiFC assays, respective constructs were cloned into expression vector
475 pICH47732 with C-terminal YFP<sup>N</sup> (NE) or YFP<sup>C</sup> (CE) by NEBuilder HiFi DNA Assembly<sup>64,67</sup>. For
476 subcellular localization studies, respective genes were cloned together with the cauliflower
477 mosaic virus 35S promoter and a mCherry or GFP tag into expression vector pICH47732<sup>64</sup>
478 using restriction enzyme digestion/ligation or NEBuilder HiFi DNA Assembly. For cell death
479 assays in *N. benthamiana* leaves GFP, AVR<sub>A13</sub>, CSEP0371 or chimera-encoding constructs were
480 cloned together with the cauliflower mosaic virus 35S promoter and a C-terminal mYFP-tag
481 derived from the pXCSG-GW-mYFP vector⁶⁶ into pICH47732⁶⁴ by restriction enzyme
482 digest/ligation. The 35S:*Mla13* construct was generated previously¹⁴. For cell death assays in
483 barley protoplasts, GFP, CSEP0371 or chimera-encoding constructs were cloned from
484 pDONR221 into pIPKb002⁶⁵ using LR Clonase II (Thermo Scientific). *pZmUbi:lysiferase*, *Mla13*
485 (in pIPKb002) and *AVR*<sub>a13</sub> (in pIPKb002) constructs were generated previously¹⁴,²⁰.

486 Generation of transgenic plants

487 The floral dip method⁶⁸ was used to generate transgenic *A. thaliana* plants and homozygous
488 single copy transgenic lines were generated base on segregation. Transgenic barley lines were
489 generated by *Agrobacterium tumefaciens*-mediated transformation of immature embryos
490 derived from *Hordeum vulgare* ssp. *vulgare* (barley) cultivar Golden Promise Fast⁶⁹ as
491 described previously⁷⁰. For this, the strain *A. tumefaciens* AGL1 carrying pIPKb002 vectors
492 with *BirA-4myc* or respective *AVR*A-*BirA-4myc constructs were used²⁰. Transformants were
493 selected on hygromycin and the presence of transgenes was confirmed by PCR using a
494 pIPKb002 forward (5'- CACCCTGTTGTTTGGTGTTACTTC -3') and BirA-specific reverse primers
495 (5'-TCGATCACAGGAAGAACAGCAAC-3') according to Edwards & Thompson 1991⁶³. Effector
496 protein expression was determined by anti-Myc western blot analysis of respective leaf
497 extracts. T2 (*AVR*<sub>A13</sub>-BirA-4myc #31-1 and #44-6, BirA-4myc #32-1 and #32-2) and respective
498 T3 lines were generated (*AVR*<sub>A13</sub>-BirA-4myc #52 and #55, BirA-4myc #53 and #54) from two
499 independent transformants (T1) of *AVR*<sub>A13</sub>-BirA-4myc and BirA-4myc, respectively.

500 Determination of late root growth responses

501 For surface sterilization, Col-0 and *AVR*<sub>a13</sub>-expressing *A. thaliana* seeds that had been
502 produced under uniform growth conditions at the same time were placed for one hour in
opened 1.5-mL tubes in a sealed box containing chlorine gas generated from 130 mL of 10%
sodium hypochlorite and 3.5 mL of 37% hydrochloric acid. Seeds were then sown on plates
containing KH₂PO₄ at 100 µM, MgSO₄ 7H₂O at 100 µM, K₂SO at 25 µM, CaCl₂ 2xH₂O at 25 µM,
KCl at 50 µM, H₃BO₃ at 30 µM, MnSO₄, H₂O at 5 µM, ZnSO₄,5H₂O at 1 µM, CuSO₄,5H₂O at 1
µM, (NH₄)₆Mo₇O₂₄ at 0.1 µM, NH₄NO₃ at 1 mM, Na-Fe-EDTA at 50 µM, MES at 250 µM and
Agar type A (A4550, Sigma Aldrich) at 0.8% with pH adjusted at 5.7 with KOH. Stratification
was performed in the dark at 4 °C for 2-3 days. Five days after growth under standard
condition, plates were transferred to identical media described above either with Iron (50 µM
FeEDTA, +Fe) or without iron (0 µM Na-Fe-EDTA, + 100 µM Ferrozine, -Fe). For this, six plants
per genotype were transferred to four 12 × 12-cm plates in a pattern in which the positions
of the genotypes were alternating in a block design (top left, top right, bottom left and bottom
right). After transfer, the plates were scanned every day for three days using the EPSON
flatbed scanners (Perfection V600 Photo, Seiko Epson CO., Nagano, Japan). Quantification
was performed by stacking the images of each day using a macro in Fiji (Macro_Match_Align)
and the root length for every day per genotype in each condition to evaluate the root growth
rate in Fiji using the segmented line. Then the mean of the root growth rate for each day and
line was calculate. These values were used to calculate the root growth rate per day for each
plant. Then, the root growth rate was divided per day under -Fe for each plant by the mean
root growth rate per day to the +Fe media for the entire related genotype. This ratio was used
as the late root growth response to low iron levels as described³⁶.

Transient transformation of barley leaf epidermal cell using particle bombardment

Transient transformation of single epidermal cells of barley cultivar Golden Promise leaves
was achieved by particle bombardment as previously described<sup>71</sup>, with following
adjustments: Primary leaves of 7 day-old-barley seedlings were placed on 1% plant agar (Duchefa Biochemie) containing 20 µg/ml Benzimidazole (Sigma-Aldrich, 194123) and bombarded with DNA-coated 1 µm gold particles (Bio-Rad) in a Biolistic Particle Delivery System (PDS-1000/He, Bio-Rad) with a helium pressure of 900 psi. Three leaves were used per plate/shot. Gold particles were used at a concentration of 60 mg/ml and coated with 6000 ng DNA/construct by using 2.5 M CaCl₂ (Roth) and 1 mg/ml Protamine (Sigma-Aldrich, A4718). Leaves were inoculated with *Bh* isolate K1 at 24 hours after particle bombardment. Samples were subjected to confocal microscopy at 60 hours after particle bombardment of barley leaf epidermal cells, which refers to 36 hours after *Bh* infection.

## Confocal microscopy

Fluorescent signals were visualized by laser scanning confocal microscopy (LSCM) using a TCS SP8 confocal microscope (Leica). GFP signal was excited with an Argon laser at a wavelength of 488 nm and emitted light was detected at 500-520 nm. mYFP signal was elicited with an Argon laser at a wavelength of 514 nm and detected at 520-540 nm. mCherry signal was excited with a 561 DPSS laser at a wavelength of 561 and emission was detected at 590 to 630 nm.

## Proximity-dependent protein labelling using the BirA/biotin system

Barley plants were grown a 16-hour light/ eight-hour dark photoperiod for four weeks. Leaves were then infiltrated with 250 µM biotin solution in 10 mM MgCl₂. After 36 h, leaf samples (300 mg per tube with 2 × 5 mm glass beads) were collected and immediately frozen in liquid nitrogen. For Strep-Tactin enrichment, frozen leaf samples were ground three times for one minute at 20 Hz with samples returned to liquid nitrogen between grinding cycles. SDT lysis buffer (prepared as 10 mL 10% SDS, 2.5 mL 1 M DTT, 2.5 mL 1 M Tris-HCl, pH 7.5) was pre-
heated to 95°C. A total of 550 µL of hot SDT buffer was added to each frozen powder sample, immediately inverted and vortexed. Samples were incubated for 5 min at 95°C and briefly centrifuged at 2000 × g, followed by sonication for 10 min. Lysates were centrifuged for 10 min at 13000 × g, and 500 µL of the supernatant was transferred to a fresh tube. A 10 µL aliquot was diluted with 40 µL PBS and mixed with 12.5 µL 5× SDS loading buffer for western blot analysis (total protein). To deplete biotin, 666 µL methanol was added to 500 µL supernatant, followed by 166 µL chloroform. Samples were vortexed and then mixed with 300 µL Milli-Q water. After centrifugation for 10 min at 2000 × g, both upper and lower phases were discarded, and the pellet was retained. The pellet was washed twice with 600 µL methanol, each time vortexed and sonicated for 10 min to break up the pellet, then centrifuged for 10 min at 13000 × g. Supernatant was completely removed and the pellet was air-dried for up to 5 min. The pellet was resuspended in 500 µL SDT buffer, vortexed, sonicated for 10 min, and vortexed again. Samples were incubated for 30 min at room temperature with shaking on an Eppendorf Thermomixer at 1000 rpm, then diluted to 4 mL in PBS buffer to achieve a final SDS concentration of 0.5%. A 50 µL aliquot was mixed with 12.5 µL 5× SDS loading buffer for western blot analysis (biotin-depleted sample). Strep-Tactin beads (50 µL slurry per sample Thermo Fisher, 20347) were washed three times with 1 mL PBS (centrifugation for 2 min at 2000 × g) and resuspended in 100 µL PBS. Fifty microliters of bead slurry were added to each protein sample, and the mixture was incubated overnight at 4°C in a 15 mL tube with gentle mixing. Beads were collected by centrifugation for 3 min at 2000 × g, and 50 µL of the supernatant was taken for western blot analysis (supernatant fraction). The beads were then washed with 2 mL PBS (pH 7.2) containing 2% SDS, centrifuged for 3 min at 2000 × g, and the supernatant was removed. The wash step was repeated six times with 10 mL PBS. Washed beads were resuspended in 1 mL PBS, transferred to a 1.5 mL
microcentrifuge tube, and centrifuged for 3 min at 2000 × g. Beads were finally resuspended in 200 µL PBS. 20 µL of bead slurry was mixed with 20 µL of 2× SDS loading buffer containing 20 mM biotin. Samples were centrifuged for 3 min at 2000 × g, and the supernatant was carefully removed. The remaining beads were stored at -80°C and subsequently subjected to Liquid Chromatography-Mass Spectrometry analysis.

## Liquid Chromatography-Mass Spectrometry

Proteins from Strep-Tactin beads were submitted to an on-bead digestion. Beads were resuspended in 25 µL digestion buffer 1 (50 mM Tris-HCl, pH 7.5; 2 M urea; 1 mM DTT; 5 ng/µL trypsin) and incubated for 30 min at 30°C in a Thermomixer (400 rpm). Beads were then pelleted by centrifugation, and the supernatant was transferred to a fresh tube. Next, 50 µL digestion buffer 2 (50 mM Tris-HCl, pH 7.5; 2 M urea; 5 mM CAA) was added to the beads. After mixing, the beads were pelleted again, and the supernatant was combined with the previous fraction. The combined supernatants were incubated overnight at 32°C in a Thermomixer (400 rpm) protected from light. Digestion was terminated by adding 1 µL trifluoroacetic acid (TFA), and peptides were desalted using C18 Empore disk membranes following the StageTip protocol⁷². Dried peptides were re-dissolved in 2% ACN, 0.1% TFA (10 µL) and diluted to 0.1 µg/µL for analysis. Samples were analyzed using an EASY-nLC 1200 (Thermo Fisher Scientific) coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). Peptides were separated on 16 cm frit-less silica emitters (New Objective, 75 µm inner diameter), packed in-house with reversed-phase ReproSil-Pur C18 AQ 1.9 µm resin. Peptides were loaded on the column and eluted for 115 min using a segmented linear gradient of 5% to 95% solvent B (0 min : 5%B; 0-5 min -> 5%B; 5-65 min -> 20%B; 65-90 min -> 35%B; 90-100 min -> 55%; 100-105 min -> 95%, 105-115 min -> 95%) (solvent A 0% ACN, 0.1%
FA; solvent B 80% ACN, 0.1%FA) at a flow rate of 300 nL/min. Mass spectra were acquired in data-dependent acquisition mode with a TOP15 method. MS spectra were acquired in the Orbitrap analyzer with a mass range of 300–1750 m/z at a resolution of 70000 FWHM and a target value of $3 \times 10^6$ ions. Precursors were selected with an isolation window of 1.3 m/z. HCD fragmentation was performed at a normalized collision energy of 25. MS/MS spectra were acquired with a target value of $10^5$ ions at a resolution of 17500 FWHM, a maximum injection time (max.) of 55 ms and a fixed first mass of m/z 100. Peptides with a charge of +1, greater than 6, or with unassigned charge state were excluded from fragmentation for MS², dynamic exclusion for 30s prevented repeated selection of precursors. Alternatively, Samples were analyzed using an Ultimate 3000 RSLC nano (Thermo Fisher) coupled to an Orbitrap Exploris 480 mass spectrometer equipped with a FAIMS Pro interface for Field asymmetric ion mobility separation (Thermo Fisher). Peptides were pre-concentrated on an Acclaim PepMap 100 pre-column (75 µM x 2 cm, C18, 3 µM, 100 Å, Thermo Fisher) using the loading pump and buffer A** (water, 0.1% TFA) with a flow of 7 µL/min for 5 min. Peptides were separated on 16 cm frit-less silica emitters (New Objective, 75 µm inner diameter), packed in-house with reversed-phase ReproSil-Pur C18 AQ 1.9 µm resin (Dr. Maisch). Peptides were loaded on the column and eluted for 130 min using a segmented linear gradient of 5% to 95% solvent B (0 min : 5%B; 0-5 min -> 5%B; 5-65 min -> 20%B; 65-90 min ->35%B; 90-100 min -> 55%; 100-105 min ->95%, 105-115 min ->95%, 115-115.1 min -> 5%, 115.1-130 min ->5%) (solvent A 0% ACN, 0.1% FA; solvent B 80% ACN, 0.1%FA) at a flow rate of 300 nL/min. Mass spectra were acquired in data-dependent acquisition mode with a TOP_S method using a cycle time of 2 seconds. For field asymmetric ion mobility separation (FAIMS) two compensation voltages (-45 and -60) were applied, the cycle times for both compensation voltages were set to 1 second. MS spectra were acquired in the Orbitrap analyzer with a mass range of 320–1200
m/z at a resolution of 60 000 FWHM and a normalized AGC target of 300%. Precursors were filtered using the MIPS option (MIPS mode = peptide), the intensity threshold was set to 5000. Precursors were selected with an isolation window of 1.6 m/z. HCD fragmentation was performed at a normalized collision energy of 30%. MS/MS spectra were acquired with a target value of 75% ions at a resolution of 15,000 FWHM, at an injection time of 120 ms and a fixed first mass of m/z 120. Peptides with a charge of +1, greater than 6, or with unassigned charge state were excluded from fragmentation for MS². The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD071751⁷³. Raw data were processed using MaxQuant software (version 1.6.3.4, http://www.maxquant.org/) with label-free quantification (LFQ) and iBAQ enabled⁷⁴,⁷⁵. MS/MS spectra were searched by the Andromeda search engine against a combined database containing the sequences from barley (Barley_Morex_V2_geneAnnotation_PGSB.all.aa⁷⁶) and sequences of 248 common contaminant proteins and decoy sequences. Trypsin specificity was required and a maximum of two missed cleavages allowed. Minimal peptide length was set to seven amino acids. Carbamidomethylation of cysteine residues was set as fixed, oxidation of methionine and protein N-terminal acetylation as variable modifications. Peptide-spectrum-matches and proteins were retained if they were below a false discovery rate of 1%. Statistical analysis of the MaxLFQ values was carried out using Perseus (version 1.5.8.5, http://www.maxquant.org/). Quantified proteins were filtered for reverse hits and hits "identified by site" and MaxLFQ values were log2 transformed. After grouping samples by condition only those proteins were retained for the subsequent analysis that had 2 valid values in one of the conditions. The relative amounts of each identified protein across samples were quantified by using label-free quantification (LFQ), and protein abundances
644 within a sample were quantified using intensity-based absolute quantification (iBAQ)⁷⁵. The
645 data were analyzed by a t-test-based analysis (LFQ data) and volcano plot analysis (iBAQ data).
646 For t-test-based analysis the enrichment of proteins between wild-type or BirA control and
647 the AVR<sub>A</sub>-BirA samples, we used the raw data (≥2 valid LFQ values) to calculate two-sample t-
648 tests with a permutation-based false discovery rate (FDR) of 5%. The enriched proteins were
649 designated as interactors of the AVR<sub>AS</sub>, respectively, if they had a p-value <0.05 and log2 fold
650 change >1 over the wild-type and BirA-4myc controls.

651 For volcano plot analysis, quantified proteins were grouped by condition and only those hits
652 were retained that had 3 valid values in one of the conditions. Missing values were imputed
653 from a normal distribution (1.8 downshift, separately for each column). Volcano plots were
654 generated in Perseus using an FDR of 5% and an S0=1. The Perseus output was exported and
655 further processed using excel (Supplementary Data 1).

656 **Barley protoplast-based cell death assay**

657 Quantification of cell death of barley mesophyll protoplasts using luciferase activity as a proxy
658 for cell viability was performed as described¹⁴ with the following three modifications: 1. A GFP
659 encoding construct in pIPKb002 was used instead of the empty vector. 2. For each
660 transfection of 300 µL of protoplasts, the plasmid solutions with a total volume of 30 µL
661 containing 10 mg of the *luciferase* reporter plasmid, 10 mg of the *Mla13* construct, and 5 mg
662 of the GFP or the respective effector construct. 3. Luciferase activity was determined at 1
663 seconds/well using a Tecan Plate Reader (TECAN, Infinite® 200 PRO).

664 *Agrobacterium*-mediated transformation of *Nicotiana benthamiana* leaves
Agrobacterium tumefaciens GV3101 pMP90K was freshly transformed by electroporation with respective constructs of interest unless specified otherwise. For each construct, 10 ml of Luria Bertani (LB) Lennox containing appropriate antibiotics were inoculated with three single colonies for each construct, and cultured over night at 28°C, 200 rpm shaking to a maximum OD₆₀₀ = 1.5. Bacteria were harvested by centrifugation at 2500 x g for 10 min and resuspended in infiltration buffer (10 mM MES, pH 5.6, 10 mM MgCl₂, and 200 mM acetosyringone) to the desired final OD₆₀₀ (OD₆₀₀ = 1 if not stated otherwise). Bacterial suspensions were incubated two to four hours at 28°C at 180 rpm, mixed equally and subsequently infiltrated into the adaxial site of leaves of three to four-week-old *N. benthamiana* plants with a needleless syringe.

## Cell death assay in Nicotiana benthamiana leaves

A. tumefaciens carrying Mla13 or the silencing inhibitors p19 or CMV2b were not freshly transformed but streaked from glycerol stocks to LB Lennox agar plates and grown as described above. Suspensions of Mla13, p19 and CMV2b-carrying bacteria was adjusted to OD₆₀₀ = 2, while bacteria freshly transformed with effector constructs were adjusted to an OD₆₀₀ = 2.4. Bacterial suspensions were then mixed at equal ratios and infiltrated to N. benthamiana leaves. At three days post infiltration cell death phenotypes were assessed and leaves were harvested for protein expression analysis.

## Co-Immunoprecipitation assay

*Nicotiana benthamiana* leaf tissue (300 mg) co-expressing the constructs of interest was harvested three days post infiltration and placed in two mL safe-lock tubes containing two 5 mm glass beads and immediately frozen in liquid nitrogen. Frozen tissue was ground to a fine powder using a Retsch Beat Beater (2 × 1 min at 30 Hz) and thereafter resuspended in 1.5 mL
688 ice-cold protein lysis buffer (150 mM Tris-HCl, pH 7.5; 150 mM NaCl; 10% glycerol; 2 mM
689 EDTA; 6 mM DTT; 0.5% Triton X-100; 1 mM PMSF) supplemented with cOmplete™ Protease
690 Inhibitor Cocktail (Roche). Samples were rotated at 4°C for 30 min to extract total proteins.
691 Protein extracts were centrifuged twice at 16,000 × g for 10 min, and the supernatant was
692 collected as the input fraction. An aliquot of the input was retained for immunoblot analysis.
693 For immunoprecipitation, 5 µL of washed GFP-Trap magnetic beads (Chromotek) were added
694 to each protein extract and incubated on a rolling wheel at 4°C for 3h. Beads were
695 magnetically separated and washed three times with 1 mL wash buffer. Bound proteins were
696 eluted by boiling the beads in 2x SDS loading buffer for subsequent immunoblotting.

697 Split-luciferase complementation (split-LUC) assay

698 Split-LUC assays in *N. benthamiana* were performed as described<sup>21</sup>. For this, *N. benthamiana*
699 leaves expressing the constructs of interest were harvested three days post-infiltration and
700 sprayed with 1 mM D-luciferin (Promega, E1602). After incubation for 10 min in the dark, the
701 luminescence signals of leaves from at least three independent plants were detected using a
702 CCD imaging system (ChemiDoc, Bio-Rad).

703 Bimolecular Fluorescence Complementation (BiFC) assay

704 BiFC assays in *N. benthamiana* were performed as described<sup>67</sup>. *N. benthamiana* leaves
705 expressing the constructs of interest were harvested two days post-infiltration and YFP
706 complementation was determined by confocal laser scanning microscopy.

707 Western blot analysis on plant-derived material

708 For analysis of protein levels and protein stability upon transgene expression, leaf material
709 from barley, *A. thaliana* and *N. benthamiana* was harvested and frozen in liquid nitrogen. Leaf
material was ground to a fine powder using pre-cooled adapters in a bead beater (Retsch) at 2 min, 30 Hz. At a ~ 100 mg fresh weight / 300 µL buffer ratio<sup>77</sup>, ground tissue was submerged in plant protein extraction buffer (150 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM EDTA, 6 mM DTT, 10% (v/v) glycerol, 1 mM PMSF, 1.5% (v/v) plant protease inhibitor cocktail (Sigma)) and subsequently centrifuged at 16,000 × g at 4°C, 10 min. The supernatant was transferred to a fresh 2 ml tube and centrifuged at 4°C for 30 min, 16,000 × g. Extracts were diluted with 4x Laemmli buffer (Bio-Rad, 1610747) containing 10% β-mercaptoethanol and heated to 95°C for 10 min. Samples were centrifuged at 16,000 × g for 10 min prior to separation by SDS-polyacrylamide-gel electrophoresis (PAGE) (Bio-Rad), and blotted onto polyvinylidene fluoride (PVDF) membranes (Merck). Membranes were blocked in TBST with 3% non-fat milk and probed with either Strep-HRP (Sigma-Aldrich, S2438) or FLAG-HRP (Sigma-Aldrich, A8592), anti-GFP (abcam, ab6556), anti-LUC (Sigma-Aldrich, L0159), anti-HA (Sigma-Aldrich, 11867423001), anti-Myc (abcam, ab9106) or anti-mCherry (Thermo Fisher, 600-401-P16S) followed by anti-rabbit IgG-HRP (Santa Cruz Biotechnology, sc-2357), anti-rat IgG-HRP (Sigma-Aldrich, A5795), or anti-mouse IgG-HRP (Santa Cruz Biotechnology, sc-2005) secondary antibody. TBST washed membranes were analyzed using the SuperSignal™ West Pico PLUS (Thermo Fisher, 34580) and SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher, 34095) using a ChemiDoc™ MP machine (Bio-Rad).

## 728 Yeast Two-Hybrid assay

The lithium acetate method<sup>78</sup> was used to co-transform respective LexA binding domain (BD) and B42 activation domain (AD) constructs into the yeast strain *EGY4.8 p8op*. Successful transformants were selected by colony growth at 30°C on SD-UHW/Glu (2% (w/v) glucose, 0.139% (w/v) yeast synthetic drop-out medium, pH 6 without uracil, histidine, tryptophan,
0.67% (w/v) BD Difco yeast nitrogen base, 2% (w/v) Bacto agar). Three days post transformation, eight to ten colonies from each sample were combined in 200 µL of water and the concentration of yeast cells was determined, adjusted and diluted to OD₆₀₀ = 1, 0.5, 0.1, 0.01 and 0.001. Ten µL of each dilution was dropped out on SD-UHW/Gal/Raf selection plates to confirm growth (+leucine) and on SD-UHWL/Gal/Raf interaction plates (-leucine). Here, glucose was replaced by 2% galactose and 1% raffinose to enable expression of the LexA BD-fused proteins. Plates were subsequently incubated at 30°C and growth of yeast transformants was documented three days after drop-out.

## Western blot analysis using yeast material

Protein levels of the constructs of interest upon expression in yeast were determined by growing yeast transformants in liquid SD-UHW/Gal/Raf medium at 30°C, 200 rpm for ~16h. Cells were harvested by centrifugation at 1000 x g for 5 min. For extraction of total protein, yeast cells were collected in 300 µL NH₄-Acetate buffer (1 M NH₄-Acetate, 150 mM NaCl, 60 mM Tris-HCl-pH 7.5, 10 mM PMSF, 5 mM EDTA, 2% Protease-Inhibitor-Cocktail (Sigma-Aldrich, P8340) and glass beads (Sigma-Aldrich, G8772) of 300 µL in volume were added. Cells were disrupted using a Retsch Beat Beater (2 min at 30 Hz). After disruption of cells, an additional 500 µL of NH₄-Acetate buffer was added to each sample and the supernatant was transferred to a new tube. Samples were incubated on ice for 1.5 h and centrifuged at 16000 x g for 10 min. The supernatant was removed and pellets were washed with 1 ml of 1 M NaCl, centrifuged at 16000 x g for 10 min and resuspended in 200 µL Urea-SDS buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 8 M urea, 2 mM EDTA, 5% glycerol, 0.004% bromophenole blue, 1% β-mercaptoethanol). Upon an additional centrifugation step at 16000 x g for 10 minutes, the proteins from the supernatant were separated by SDS-PAGE (Bio-Rad), and blotted onto polyvinylidene fluoride (PVDF) membranes (Merck). Membranes were blocked in TBST with
757 3% non-fat milk and probed with either anti-HA (Roche 11867423001 for B42-HA fusion
758 proteins) or anti-LexA (Santa Cruz Biotechnology sc7544 for LexA-fusion proteins) primary
759 antibodies followed by anti-rat (Sigma-Aldrich, A5795) or anti-mouse IgG-HRP (Santa Cruz
760 Biotechnology, sc2005) secondary antibodies as appropriate. TBS-T washed membranes were
761 analyzed using the SuperSignal™ West Pico PLUS (Thermo Fisher, 34580) and SuperSignal™
762 West Femto Maximum Sensitivity Substrate (Thermo Fisher, 34095) using a ChemiDoc™ MP
763 machine (Bio-Rad).
764

764

765

Acknowledgements

766 We thank Nicolaus von Wirén and Ricardo Giehl for useful discussion on the role of plant iron
767 homeostasis during pathogen attack; Petra Bauer for suggestions on iron supplementation
768 experiments; the UoC Institute for Plant Sciences' greenhouse team for their expertise in
769 maintenance and high-yield propagation of barley; Dennis Schapelhouman and Saskia
770 Bodendorf for maintaining Bh isolate K1; Paul Schulze-Lefert for making the MPIPZ facilities
771 available for the generation of transgenic AVR<sub>a13</sub> lines; Gabriel Hacker, Sabine Haigis and
772 Emma Crean for technical assistance in propagating barley plants and *in vitro* growth of barley
773 under differential iron conditions, generating AVR<sub>a</sub>-overexpressing lines, and the selection
774 transgenic barley lines, respectively. This work was funded by the Deutsche
775 Forschungsgemeinschaft (DFG, German Research Foundation) Emmy Noether Programme
776 (SA 4093/1-1 to IMLS), the DFG's Collaborative Research Centre Grant (SFB-1403—414786233
777 to IMLS, GD), the Germany's Excellence Strategy CEPLAS (EXC-2048/1, project 390686111
778 IMLS, GD) and the Max-Planck-Society (HN).
779

Data availability

780

The mass spectrometry proteomics data have been deposited to the ProteomeXchange

781

Consortium via the PRIDE partner repository with the dataset identifier PXD071751.

782

Author contributions

783

Experiments: WS (proximity labelling, Y2H, CoIP, Split-LUC, confocal microscopy, pathogen

784

infection), MB-S (Y2H, cell death assays, confocal microscopy, pathogen infection, ROS), SCS

785

(LC-MS/MS), MPP (root growth inhibition), MH and IMLS (generation, selection, assessment

786

and propagation of transgenic plants, generation of T2 and T3 transgenic lines). Data analysis:

787

WS, MB-S, SCS, IMLS. Investigation: WS, MB-S, GD, HN, IMLS. Supervision: HN, IMLS. Funding

788

acquisition: IMLS, GD. Project administration, conceptualization and study design: IMLS.

789

Writing: WS, MBS, IMLS with input from all authors. Editing: All authors.

790

Competing interests

791

The authors declare no competing interests.

792

Figure legends

793

**Figure 1:** Proximity-dependent protein labelling identifies HvSRF3 as specific interactor of the *Blumeria hordei* (Bh) virulence factor AVR<sub>A13</sub>. a Number of barley proteins specifically identified by LC-MS/MS (Log2 FC>1; p<0.05) in proximity labelling samples from transgenic barley expressing AVR<sub>A</sub> effectors lacking respective signal peptide sequences as indicated and when compared to wild-type (WT) and BirA control samples. b Venn diagram investigating the overlap of AVR<sub>A</sub>-interacting proteins from a. *Blumeria hordei* (Bh) disease symptoms on

794

795

796

797

798
799 leaves of independent transgenic barley lines (T2) expressing *BirA* or *AVR*<sub>a13</sub>-*BirA* at five days post infection. Figures shown are representatives of three independent experiments. **d** 800 Quantification of *Bh* colonies at the adaxial and abaxial site of the infected leaves across all 801 replicates of experiments described in **c**. Differences between samples assessed by Kruskal- 802 Wallis (p=1.177e-05) and subsequent Dunn's test. Samples marked by identical letters in the 803 plots do not differ significantly (p>0.05) in the Dunn's test. **e** Volcano plot analysis of proteins 804 specifically identified by LC-MS/MS (at Log2 = 1; p>0.05) in WT (green dots) BirA control 805 (green) and *AVR*<sub>A13</sub> (red) proximity labelling samples derived from independent transgenic 806 lines as indicated. Bold dots denote peptides specific to HvSRF3. **f** Particle bombardment- 807 based expression of *HvSRF3-mYFP* in barley epidermal cells. Leaves were infected with *Bh* at 808 24 hours post particle bombardment and microscopic analysis was performed 60 hours post 809 bombardment using a Leica TCS SP8 confocal laser scanning microscope. Top image: YFP 810 channel (520-540 nm), Bottom image: Brightfield; Green dashes: Transformed cell not 811 attacked by *Bh*; Orange dashes: Transformed cell under *Bh* attack; Inf: successful *Bh* infection, 812 Pen: *Bh* penetration attempt; C: Conidiospore.

814

815 **Figure 2: Blumeria hordei (Bh) AVR<sub>A13</sub> can interact with the cytoplasmic domain (CD) of**
816 **HvSRF3.**
817 **a** Domain structure of *Arabidopsis thaliana* and barley Strubbelig Receptor Family 3
818 (SRF3).
819 **b** Phylogeny (Neighbor-Joining tree) of *A. thaliana* and barley Strubbelig Receptor
820 Family members (HvSRFs).
821 **c** and **d** *Nicotiana benthamiana* leaves were transiently
822 transformed with the constructs indicated. At three days post transformation, protein-protein
823 interaction was determined by the complementation of the luciferase (LUC) reporter by nLUC
824 and cLUC-fused protein upon addition of substrate (c) and GFP-TRAP-based
825 immunoprecipitation of GFP or mYFP tagged proteins followed by anti-GFP and anti-HA
823 western blot analysis (d). Figures shown are representatives of at least three independent
824 experiments. e Yeast cells were transformed with B42 activation domain (AD)- and HA tag-
825 fused constructs encoding GFP or AVR<sub>A13</sub> variants together with LexA binding domain (BD)-
826 fused constructs encoding MLA13, HvSRF3-L1 cytoplasmic domain (HvSRF3-L1<sup>CD</sup>) or HvSRF3-
827 L2 cytoplasmic domain (HvSRF3-L2<sup>CD</sup>). Growth of transformants was determined on selective
828 growth medium with leucine (+leucine), and interaction of proteins was determined by
829 leucine reporter activity reflected by growth of yeast on selective medium lacking leucine (-
830 leucine). Figures shown are representatives of at least three independent transformation
831 experiments and pictures were taken three days after drop out. Signal peptides were depleted
832 from all effector-encoding constructs.

833 **Figure 3: The specificity of the HvSRF3-AVR<sub>A13</sub> association in yeast is mediated by the HvSRF3**
834 **kinase domain. a and b** Yeast cells were transformed with B42 activation domain (AD)- and
835 HA tag-fused constructs encoding GFP or AVR<sub>A13</sub> variants together with LexA binding domain
836 (BD)-fused constructs encoding either the HvSRF3-L3 cytoplasmic domain (HvSRF3-L3<sup>CD</sup>) or
837 HvSRF3-L4 cytoplasmic domain (HvSRF3-L4<sup>CD</sup>) (a) or HvSRF3-L1 kinase domain (HvSRF3-L1<sup>KD</sup>)
838 or HvSRF3-L2 kinase domain (HvSRF3-L2<sup>KD</sup>) (b). **c** Domain architecture of HvSRF3 isoforms and
839 chimeric HvSRF3 constructs composed of the HvSRF3 isoform L1 (pale green), L2 (green) or L4
840 (grey) juxtamembrane domain (JM) and kinase domain (KD). **d and e** Yeast cells were
841 transformed with B42 activation domain (AD)- and HA tag-fused constructs encoding GFP or
842 AVR<sub>A13</sub> variants together with LexA binding domain (BD)-fused constructs encoding chimeric
843 HvSRF3 constructs from **c**. Growth of transformants was determined on selective growth
844 medium with leucine (+leucine), and interaction of proteins was determined by leucine
845 reporter activity reflected by growth of yeast on selective medium lacking leucine (-leucine).
Signal peptides were depleted from all effector-encoding constructs and figures shown are representatives of at least three independent transformation experiments and pictures were taken three days after drop out. **f and g** AlphaFold models of ATP and Mg²⁺- bound HvBAK1KD (pTM =0.9) (f), HvSRF3-L1KD (pTM =0.9) and HvSRF3-L2KD (pTM =0.89) (g). The N-lobe, C-lobe, P-loop, DFG motif, activation loop, catalytic loop in HvBAK1KD or respective residues in HvSRF3-L1/HvSRF3-L2KD are coloured cyan, pale green, orange, blue, red and yellow, respectively. **h** Sequence alignment of critical kinase motifs in AtBAK1KD, HvBAK1KD, AtSRF3KD, HvSRF3-L1KD, HvSRF3-L2KD, HvSRF3-L3KD, HvSRF3-L4KD. Residues involved in phosphotransferase activity are highlighted in bold.

**Figure 4: AVR<sub>A13</sub> affects HvSRF3 protein localization and interaction with HvBAK1.** a Yeast cells were transformed with B42 activation domain (AD)- and HA tag-fused constructs encoding the HvSRF3<sup>CD</sup> together with the LexA binding domain (BD)-fused construct encoding the cytoplasmic domain of HvBAK1 (HvBAK1<sup>CD</sup>). Growth on selective medium with leucine (+leucine) confirmed transformation, while growth on medium lacking leucine (-leucine) indicated protein-protein interaction *via* activation of the leucine reporter. Photographs were taken three days after drop-out. **b** *N. benthamiana* leaves were transiently co-transformed with C-terminally nLUC-fused and C-terminally cLUC-fused constructs as indicated. At three days post transformation, protein-protein interaction was determined by the complementation of the luciferase (LUC) reporter by chemiluminescence upon addition of substrate. Pictures shown are representative results from at least three independent biological replicates. **c** *Nicotiana benthamiana* leaves were transiently co-transformed with C-terminally YFP<sup>N</sup>-fused and C-terminally YFP<sup>C</sup>-fused constructs as indicated. At two days post transformation, protein-protein interaction was determined by the complementation of the
YFP fluorescence signals using a Leica TCS SP8 confocal laser scanning microscope. Pictures shown are representative results from at least three independent biological replicates. Scale bars: 25 µm. **d** *N. benthamiana* leaves were transiently co-transformed with constructs encoding GFP or mYFP tagged effector constructs as indicated together with C-terminally nLUC-fused HvSRF3 and C-terminally cLUC-fused HvBAK1 constructs. At three days post transformation, protein-protein interaction was determined by the complementation of the luciferase (LUC) reporter by chemiluminescence upon addition of substrate. Pictures shown are representative results from at least three independent biological replicates. **e** Quantification of luciferase signals across all replicates of experiments described in d. Differences between samples assessed by Kruskal-Wallis (p=1.324e-05) and subsequent Dunn's test. Samples marked by identical letters in the plots do not differ significantly (p>0.05) in the Dunn's test. **f** *N. benthamiana* leaves were transiently transformed with constructs encoding mCherry or AVR<sub>A13</sub>-mCherry together with C-terminally YFP<sup>N</sup>-fused HvSRF3 and C-terminally YFP<sup>C</sup>-fused HvBAK1 constructs as indicated. At two days post transformation, protein-protein interaction was determined by complementation of the YFP fluorescence signal using a Leica TCS SP8 confocal laser scanning microscope. Pictures shown are representative results from at least three independent biological replicates. Scale bars: 25 µm. **g** Quantification of luciferase signals across all replicates of experiments described in f. Differences between samples assessed by Kruskal-Wallis (p=3.015e-08) and subsequent Dunn's test. Samples marked by identical letters in the plots do not differ significantly (p>0.05) in the Dunn's test. **h** *N. benthamiana* leaves were transiently transformed with constructs encoding HvSRF3-mCherry together with constructs encoding GFP, AVR<sub>A13</sub>-mYFP, AVR<sub>A13</sub>-V2-mYFP or CSEP0371-mYFP. GFP/YFP and mCherry fluorescence signals were determined using a Leica TCS SP8 confocal laser scanning microscope two days post transformation. Pictures
shown are representative results from at least three independent biological replicates. Scale
bars: 25 µm. i Quantification of mCherry signals across all replicates of experiments described
in h. j Quantification of GFP/YFP signals across all replicates of experiments described in h.
Differences between samples were assessed by Kruskal-Wallis (p= 2.2e-16 for I and p=
0.09609 for J) and subsequent Dunn's test. Samples marked by identical letters in the plots
do not differ significantly (p>0.05) in the Dunn's test. n.s. = no significant difference. Signal
peptides were depleted from all effector-encoding constructs throughout.

**Figure 5:** *AVR<sub>A13</sub> can associate with *Arabidopsis thaliana* SRF3 (AtSRF3) and modulates plant responses to differential iron levels in *A. thaliana* and barley. a and b *Nicotiana benthamiana* leaves were transiently transformed with the constructs indicated. At three days post transformation, protein-protein interaction was determined by the complementation of the luciferase (LUC) reporter by chemiluminescence upon addition of substrate (a) and GFP-TRAP-based immunoprecipitation of GFP or mYFP tagged proteins followed by anti-GFP and anti-HA western blot analysis (b). Figures shown are representatives of at least three independent experiments. c Yeast cells were transformed with B42 activation domain (AD)- and HA tag-fused constructs encoding GFP or *AVR<sub>A13</sub>* variants together with LexA binding domain (BD)-fused constructs encoding the AtSRF3 cytoplasmic domain (AtSRF3<sup>CD</sup>). Growth of transformants was determined on selective growth medium with leucine (+leucine), and interaction of proteins was determined by leucine reporter activity reflected by growth of yeast on selective medium lacking leucine (-leucine). Figures shown are representatives of at least three independent transformation experiments and pictures were taken three days after drop out. d *A. thaliana* rosettes of Col-0 and *AVR<sub>a13</sub>*-expressing lines grown under standard conditions. e Root length of *A. thaliana* lines from d at eight days grown *in vitro* under iron
sufficient growth conditions. f Late root growth response (Platre *et al.*, 2024) of Col-0 wild-type and *AVR*<sub>a13</sub>-expressing *A. thaliana* lines grown for four days with (50 µM FeNaEDTA) or without iron (-FeFZ100). Differences between lines were assessed by analysis of variance (ANOVA p=1.75E-05) and subsequent Tukey post hoc test. Samples marked by identical letters in the plots do not differ significantly (p>0.05) in the Tukey test. Values were obtained from three independent experiments. g and h Leaves of barley cultivar Golden Promise Fast plants were pre-treated with 0.5 g/L FeEDDHA or water (mock) prior to infection with *Blumeria hordei* (*Bh*). Fungal growth was determined at five days post infection. Figures shown are representatives of at least three independent experiments (h) and quantification of *Bh* colonies was performed from pictures taken of the adaxial and abaxial site of the infected leaves across all replicates of experiments described (g). Differences between treatments were assessed by Kruskal-Wallis test (p=1.352e-05). i and j *Bh* disease symptoms on leaves of independent transgenic barley lines (T3) expressing *BirA* or *AVR*<sub>a13</sub>-*BirA* at five days post infection upon treatment with water (mock) or 0.5 g/L FeEDDHA. Figures shown are representatives of at least three independent experiments (i). Quantification of *Bh* colonies (j) was performed from pictures taken of the adaxial and abaxial site of the infected leaves across all replicates of experiments described in i. Differences between treatments were assessed by Kruskal-Wallis test (p<2.2e-16) and subsequent Dunn's test. Samples marked by identical letters in the plots do not differ significantly (p>0.05) in the Dunn's test. Signal peptides were depleted from all effector-encoding constructs throughout.

<caption>**Figure 6:** A central loop-containing region in *AVR*<sub>A13</sub> specifies HvSRF3 interaction and MLA13 activation. **a** Sequence alignment of *Blumeria hordei* CSEP protein family 34 members *AVR*<sub>A13</sub> (CSEP0372), CSEP0371, CSEP0373 and CSEP0374. **b** Structural overlay of *AVR*<sub>A13</sub> (CSEP0372,</caption>
939 PDB 9FYC) with AlphaFold models of CSEP0371 (pTM=89.2), CSEP0373 (pTM=86.2) and
940 CSEP0374 (pTM=87.2). c Yeast 2-hybrid assay with cells transformed with B42 activation
941 domain (AD)- and HA tag-fused constructs encoding GFP or effectors together with LexA
942 binding domain (BD)-fused constructs encoding the cytoplasmic domain of HvSRF3
943 (HvSRF3CD). Growth of transformants was determined on selective growth medium with
944 leucine (+leucine), and interaction of proteins was determined by leucine reporter activity
945 reflected by growth of yeast on selective medium lacking leucine (-leucine). Figures shown are
946 representatives of at least three independent transformation experiments and pictures were
947 taken three days after drop out. d Chimeras composed of AVR<sub>A13</sub> aa residues (orange) and
948 CSEP0371 aa residues (blue) as indicated. Vertical lines indicate differential amino acid
949 residues between AVR<sub>A13</sub> and CSEP0371. e Yeast cells were transformed with B42 AD- and HA
950 tag-fused constructs encoding GFP, AVR<sub>A13</sub>, CSEP0371 or chimeras together with HvSRF3<sup>CD</sup>
951 fused to the LexA BD. Protein interaction was determined as described in c. f GFP or C-
952 terminally mYFP-fused AVR<sub>A13</sub>, CSEP0371 or chimeras were co-overexpressed with Mla13-myc
953 in leaves of four-week-old Nicotiana benthamiana plants using Agrobacterium-mediated
954 transformation. Cell-death was determined three days post transformation. g Mesophyll
955 protoplasts of barley cultivar Golden Promise were co-transfected with a pZmUBQ:lysiferase
956 reporter plasmid, Mla13, and constructs carrying GFP, CSEP0371, AVR<sub>a13</sub> or chimeras all in
957 pIPKb002. Luciferase activity was determined at 16 h post transfection and normalized to the
958 respective GFP sample. Values were obtained from at least four independent transfections
959 performed on at least three independent days. Differences between samples processed
960 simultaneously were assessed by analysis of variance (ANOVA p ≤ 2.3e-06) and subsequent
961 Tukey post hoc test. Samples marked by identical letters in the plots do not differ significantly
962 (p>0.05) in the Tukey test. Signal peptides were depleted from all effector-encoding
963 constructs (c, e, f, g). **h** AVR<sub>A13</sub> structure (PDB 9FYC) indicating the central region (aa 44-70)

964 required for HvSRF3 binding and MLA13 activation. **i** Structural arrangement of AVR<sub>A13</sub> (PDB

965 9FYC), CSEP0371 (pTM<sub>AVRA13</sub>=89.2) and CSEP0374 structure (pTM<sub>AVRA13</sub>=87.2). Central region

966 required for HvSRF3 binding and MLA13 activation in orange (h, i).

967

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<caption>Fig. 1</caption>

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b. Copyright preprint doi: https://doi.org/10.64898/2026.01.08.698370; this version posted January 9, 2026. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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bioRxiv preprint doi: https://doi.org/10.64898/2020.0108880

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is the copyright holder for this version and the terms of use under which it may be used.

aa 289

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bioRxiv preprint doi: https://doi.org/10.64898/2026.011.08.698370; this version posted January 9, 2026. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
<figure><img src="image_19.png" /><figcaption>Fig. 5</figcaption></figure>
<figure><img src="image_20.png" /><figcaption>Fig. 6</figcaption></figure>

bioRxiv preprint doi: https://doi.org/10.64898/2026.01.08.698370; this version posted January 9, 2026. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.