A set of Arabidopsis genes involved in the accommodation of the downy mildew pathogen Hyaloperonospora arabidopsidis

The intracellular accommodation structures formed by plant cells to host arbuscular mycorrhiza fungi and biotrophic hyphal pathogens are cytologically similar but it remains unclear whether these interactions build on an overlapping genetic framework. In legumes, the malectin-like domain leucine-rich repeat receptor kinase SYMRK, the cation channel POLLUX and members of the nuclear pore NUP107-160 subcomplex are essential for symbiotic signal transduction and arbuscular mycorrhiza development. Here we identified members of these three groups in Arabidopsis thaliana and explored their impact on the interaction with the oomycete downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). We report that mutations in the corresponding genes reduced the reproductive success of Hpa as determined by sporangiophore and spore counts. We discovered that a developmental transition of haustorial shape occurred significantly earlier and at higher frequency in the mutants. Analysis of the multiplication of extracellular bacterial pathogens, Hpa-induced cell death or callose accumulation, as well as Hpa- or flg22-induced defence marker gene expression, did not reveal any traces of constitutive or exacerbated defence responses. These findings point towards an overlap between the plant genetic toolboxes involved in the interaction with biotrophic intracellular hyphal symbionts and pathogens in terms of the gene families involved. Author Summary Our work reveals genetic commonalities between biotrophic intracellular interactions with symbiotic and pathogenic hyphal microbes. The majority of land plants engages in arbuscular mycorrhiza (AM) symbiosis with phosphate-acquiring arbuscular mycorrhizal fungi to avoid phosphate starvation. Nutrient exchange in this interaction occurs via arbuscules, tree-shaped fungal structures, hosted within plant root cells. A series of plant genes including the Symbiosis Receptor-like kinase (SYMRK), members of the NUP107-160 subcomplex and nuclear envelope localised cation channels are required for a signalling process leading to the development of AM. The model plant Arabidopsis thaliana lost the ability to form AM. Although the ortholog of SYMRK was deleted during evolution, members of the malectin-like domain leucine-rich repeat receptor kinases (MLD-LRR-RKs) gene family, components of the NUP107-160 subcomplex, and an ortholog of the nuclear envelope-localized cation channel POLLUX, are still present in the Arabidopsis genome, and Arabidopsis leaf cells retained the ability to accommodate haustoria, presumed feeding structures of the obligate biotrophic downy mildew pathogen Hyaloperonospora arabidopsidis. We discovered that both of these plant-microbe interactions utilize a corresponding set of genes including the ortholog of POLLUX, members of the NUP107-160 subcomplex and members of the MLD-LRR-RK gene family, thus revealing similarities in the plant program for the intracellular accommodation of biotrophic organisms in symbiosis and disease.

their impact on the interaction with the oomycete downy mildew pathogen Hyaloperonospora 23 arabidopsidis (Hpa). We report that mutations in the corresponding genes reduced the reproductive 24 success of Hpa as determined by sporangiophore and spore counts. We discovered that a developmental 25 transition of haustorial shape occurred significantly earlier and at higher frequency in the mutants. similarities between accommodation structures for intracellular microbes raised the hypothesis that the 59 corresponding symbiotic and pathogenic associations rely on a similar or even overlapping genetic 60 program [4]. This would imply that filamentous hyphal pathogens exploit an Achilles heel, the presence 61 of the symbiotic program in most land plant species, for their own parasitic lifestyle [11]. 62 lower in Hpa-infected shrk1 x shrk2 plants than in infected wild-type plants, however, it is unlikely that 158 this is the reason for the increased pathogen resistance of the double mutant. Moreover, the ability of 159 Hpa to suppress callose deposition around the haustorial neck region [46] was not disturbed in the 160 SNUPO mutants compared to the wild-type (Fig. 4 b). 161 In addition to the expression levels of defence marker genes, we investigated other symptoms typically 162 associated with deregulated immune responses. We could not observe any developmental or growth 163 defects, which are typically a result of the hyper-activation of the SA-dependent defence pathway [35], 164 in SNUPO mutants grown side-by-side with the wild-type and the dwarf mutant suppressor of npr1-1, 165 constitutive 1 (snc1 [47]) (S10 Fig.). Furthermore, we analysed constitutive and Hpa-induced cell death 166 responses in SNUPO mutants and the wild-type (Fig. 5). Most of the non-infected leaves did not display 167 any sign of cell death (Fig. 5, first column), but dark-blue stained dead cells were sporadically observed 168 with no significant differences between non-infected leaves of both wild-type and SNUPO mutants ( To examine whether mutation of SNUPO genes has an effect on signalling related to PAMP-triggered 176 immunity, we inspected the transcript levels of Flg22-induced Receptor-like Kinase 1 (FRK1 [48]) and 177 ERF1 in the wild-type, the SNUPO mutants and a fls2 mutant (Fig. 6) in response to the bacterial 178 flagellin-derived peptide flg22 [49]. Transcript levels in mock-treated samples were not different in the 179 wild-type and the analysed mutants, except for the shrk1 mutant that displayed a slight increase in basal 180 sec13 double mutants that contained slightly decreased basal levels of ERF1 transcripts. The deviations 182 observed for individual mutants are unlikely responsible for the increased Hpa resistance. Six hours after 183 flg22 treatment the tested genes were all upregulated to the same extent in the SNUPO mutants and the 184 wild-type (Fig. 6 a). Furthermore, we investigated the growth behaviour of P. syringae on the wild-type 185 and on SNUPO mutants (Fig. 6 b). P. syringae pv. tomato (Pto) DC3000 induces the activation of SA-186 dependent defence signalling in the host, and deregulation of this pathways impairs P. syringae resistance 187 [50]. The growth of Pto DC3000 wild-type or the avirulent ΔAvrPto/PtoB strain was unaltered on the A. 188 thaliana SNUPO mutants, providing further evidence that they do not exhibit constitutive or enhanced 189 activation of SA-dependent defences (Fig. 6 b). largely unexplored. Here we tested the hypothesis that the biotrophic pathogen Hpa utilises a gene set 195 related to that enabling intracellular accommodation in the context of AM symbiosis. 196 SNUPO mutants reveal a link between the timing of haustorial shape shifts and reproductive 197 success 198 We observed -based on sporangiophore and spore counts -that A. thaliana SNUPO mutants are impaired 199 in supporting the reproduction of the oomycete pathogen Hpa. This was associated with a shift in the 200 haustoria morphology. Intriguingly, we observed congruent phenotypes in mutants of diverse protein 201 classes with the only connector between these protein classes being their reported involvement in root 202 endosymbioses. The frequency of multilobed haustoria increased in the wild-type as well as in the 203 mutants over time revealing a clear connection between haustoria morphology and the age of the 204 interaction, which is in line with a previously observed continuous growth of Albugo and Hpa haustoria 205 over time (Eric Kemen and Marco Thines; independent personal communication). As the frequency of 206 multilobed haustoria was significantly higher in the A. thaliana SNUPO mutants at all time points 207 analysed, it appears that haustoria growth is accelerated in the SNUPO mutants. It has been postulated 208 that haustoria are the main avenue for nutrient acquisition from the host [51]. Considering the agricultural 209 impact of biotrophic hyphal pathogens, surprisingly little is known about the precise function of haustoria 210 and changes thereof during haustoria development. Senescence of haustoria has been associated with 211 encasement which was discussed to likely reduce their functionality [52]. Therefore, it is possible that 212 the accelerated formation of multilobed haustoria is a sign of senescence and thus directly responsible 213 for the reduction in Hpa reproductive success. This scenario would suggest a role of Arabidopsis SNUPO 214 genes in maintaining the single lobed, and presumably functional, stage of Hpa haustoria. In an opposite 215 scenario, the surface increase from single lobed to multilobed haustoria may benefit the oomycete and 216 the genes under study are involved in delaying the progress of haustoria development into the multilobed 217 stage. However, this scenario is not compatible with the reduced sporangiophore and spore count on the 218 mutants. 219

SNUPO mutants exhibit no traces of altered plant defence responses or regulation 220
Based on the analysis of a wide range of defence symptoms in the classical PTI assays and three different 221 pathosystems, we conclude that A. thaliana SNUPO mutants display unaltered levels and frequencies of 222 defence responses. Consequentially, the impairment of the Hpa interaction is not due to deregulated 223 defence in these mutants. 224 Arabidopsis SNUPO mutants are specifically impaired in the Hpa interaction 225 We were unable to detect differences in the interaction of A. thaliana SNUPO  to Arabidopsis SNUPO genes -are rather involved in general plant disease resistance than in 248 compatibility with and the accommodation of a hyphal organism. 249 It is a long-standing hypothesis that plant pathogens exploit an Achilles heel of the plant, genetic 250 pathways for the intracellular accommodation of mutualistic symbionts such as AM fungi or nitrogen-251 fixing bacteria [4,11]. Recently the oomycete pathogen Phytophthora palmivora, which forms haustoria 252 that only last for a few hours, has emerged as a hemi-biotrophic model system in two independent 253 laboratories to test this hypothesis in legume mutants. This pathogen quickly progresses to a necrotrophic 254 phase and structural alterations in haustoria development are thus less likely to affect pathogen fitness 255 It will be therefore interesting to identify the mechanistic commonalities between symbiotic and 282 pathogenic interactions with hyphal organisms that are controlled by corresponding gene sets. 283

Retention of SNUPO genes in the Arabidopsis genome 284
The loss of AM symbiosis in A. thaliana and in four other independent plant lineages was correlated with 285 the absence of more than 100 genes with potential roles in AM [29-31,69]. While the exploitation of 286 symbiotic programs by pathogens might explain the consistent deletion of CSGs from five independent 287 plant lineages, it raises the question, which evolutionary forces retained SNUPO genes in the Arabidopsis 288 genome. A housekeeping function was not revealed since no pleiotropic developmental phenotypes were 289 observed in the mutants. While, it might be possible that these genes limit Hpa colonisation, thereby 290 forcing the oomycete to sporulate earlier or more profusely, our results leave us with the unexpected 291 finding that the only detected role for the SNUPO genes in A. thaliana is the support of an oomycete. It 292 will be interesting to find out whether ecological conditions exist, under which oomycete colonization 293 might provide a selective advantage to the host plant, or whether SNUPO genes are also involved in the 294 accommodation of beneficial microbes like C. tofieldiae justifying their retention in the Arabidopsis 295 genome [70]. 296

Seed sterilization and plant growth 298
All A. thaliana mutants described in the manuscript were of Col-0 ecotype, except for ios1, which was 299 either of Col-0 or Ler ecotype. Seeds were obtained from "The Nottingham Arabidopsis Stock 300 Centre" -NASC [71]

Pathogen assays and phenotypic analyses 312
To collect spores for inoculation, A. thaliana wild-type (Col-0 or Ler) leaves with sporulating Hpa isolate 313 NoCo2 or Hpa isolate Waco9, respectively, were harvested seven days post inoculation (dpi) and placed 314 into 10 ml deionized H2O and vortexed for 2 s in 15 ml reaction tubes. The spore suspension was then 315 filtered through a Miracloth filter and sprayed onto 12-days-old plants using a spraying device. 316 Subsequently, plants were placed into trays and covered with wet translucent plastic lids. Trays were 317 sealed to maintain high humidity, and plants were grown under long day conditions (16 h light, 18°C, 85 318 µmol m -2 s -1 ). 319 seedlings per background were harvested into reaction tubes containing 1 ml deionized H2O and vortexed 321 for 3 min. Spores were counted with a Fuchs Rosenthal chamber. For sporangiophore counting and the 322 investigation of haustoria shape and penetration efficiency cotyledons or leaves were harvested and 323 stained in 0.01% trypan-blue-lactophenol for 3 min at 95°C and 5 h at room temperature, followed by 324 overnight clearing in chloral hydrate (2.5 g/ml). Samples were mounted in glycerol for subsequent 325 differential interference contrast microscopy with a Leica DMI6000B.