PacC-dependent adaptation and modulation of host cellular pH controls hemibiotrophic invasive growth and disease development by the rice blast fungus

Many of the world’s most serious crop diseases are caused by hemibiotrophic fungi. These pathogens have evolved the ability to colonize living plant cells, suppressing plant immunity responses, before switching to necrotrophic growth, in which host cells die, providing the energy to fuel sporulation and spread of the fungus. How hemibiotrophic pathogens switch between these two lifestyles remains poorly understood. Here, we report that the devastating rice blast fungus, Magnaporthe oryzae, manipulates host cellular pH to regulate hemibiotrophy. During infection by M. oryzae, host plant cells are alkalinized to pH 7.8 during biotrophic growth, but later acidified to pH 6.5 during necrotrophy. Using a forward genetic screen, we identified alkaline-sensitive mutants of M. oryzae that were blocked in biotrophic proliferation and impaired in induction of host cell acidification and necrotrophy. These mutants defined components of the PacC-dependent ambient pH signal transduction pathway in M. oryzae. We report that PacC exists as a full-length repressor, PacC559, and a truncated transcriptional activator, PacC222, which localize to the fungal nucleus during biotrophic growth and to the cytoplasm during necrotrophy. During biotrophy, PacC222 directly activates genes associated with nutrient acquisition and fungal virulence, while PacC559 represses genes associated with saprophytic mycelial growth and sporulation, which are subsequently de-repressed during necrotrophy. When considered together, our results indicate that temporal regulation of hemibiotrophy by M. oryzae requires PacC-dependent sensing and manipulation of host cellular pH. Author Summary Crop diseases caused by fungi represent some of the most serious threats to global food security. Many fungal pathogens have evolved the ability to invade living plant tissue and suppress host immunity, before switching to a completely different mode of growth, in which they are able to kill host plant cells. This lifestyle– called hemibiotrophy –is exemplified by the blast fungus, Magnaporthe oryzae, which causes devastating diseases of rice, wheat and many other grasses. We found that during infection by M. oryzae, host cells initially have an alkaline pH, when the fungus is growing in living tissue, but pH rapidly becomes acidic, as host tissue is killed. We identified mutants of the blast fungus that were sensitive to alkaline pH and this enabled us to identify the signal transduction pathway by which the fungus responds to changes in ambient pH. We found that mutants in the pH response pathway were blocked in invasive fungal growth and could not cause acidification of host tissue. Consequently, they are unable to cause blast disease. We characterized the central regulator of this pathway, the PacC transcription factor, which unusually can act as both a repressor and an activator of fungal gene expression. During biotrophic invasive growth, PacC activates many genes previously reported to be required for virulence, including several associated with nutrient acquisition, and at the same time represses genes associated with vegetative growth and sporulation. The PacC signaling pathway is therefore necessary for regulating the switch in fungal lifestyle associated with causing blast disease.

Magnaporthe oryzae, manipulates host cellular pH to regulate hemibiotrophy. During infection by 23 M. oryzae, host plant cells are alkalinized to pH 7.8 during biotrophic growth, but later acidified to 24 pH 6.5 during necrotrophy. Using a forward genetic screen, we identified alkaline-sensitive mutants 25 of M. oryzae that were blocked in biotrophic proliferation and impaired in induction of host cell 26 acidification and necrotrophy. These mutants defined components of the PacC-dependent ambient 27 pH signal transduction pathway in M. oryzae. We report that PacC exists as a full-length repressor, 28 PacC 559 , and a truncated transcriptional activator, PacC 222 , which localize to the fungal nucleus 29 during biotrophic growth and to the cytoplasm during necrotrophy. During biotrophy, PacC 222 30 directly activates genes associated with nutrient acquisition and fungal virulence, while PacC 559 31 represses genes associated with saprophytic mycelial growth and sporulation, which are subsequently 32 de-repressed during necrotrophy. When considered together, our results indicate that temporal 33 regulation of hemibiotrophy by M. oryzae requires PacC-dependent sensing and manipulation of host 34 cellular pH. 54 Plant pathogenic fungi can be broadly classified into species that always invade living host tissue, 55 called biotrophs, which evade recognition and suppress host immunity to systemically colonize host 56 plants, and necrotrophic pathogens which overwhelm plant defenses by rapidly killing plant cells, to 57 acquire nutrients from dead or dying tissue [1][2][3]. Both groups of pathogens exhibit distinct 58 characteristics in terms of the weapons they deploy to infect host plants-such as effector proteins, 59 toxins, and metabolites [4]. Host plants have, in turn, evolved distinct immune signaling pathways to 60 respond to biotrophs and necrotrophs [5]. There is, however, a third group of pathogens, 61 encompassing many of the world's most serious disease-causing fungi that exhibit both styles of 62 growth. These pathogens are known as hemibiotrophs and initially infect plants like a biotroph, 63 eliciting little response or disease symptoms in their host, but later, at a given point during infection, 64 they switch to killing cells, inducing cellular necrosis and fueling their own sporulation [1]. However, 65 the mechanism by which hemibiotrophic fungal pathogens switch between biotrophy and 66 necrotrophy remains poorly understood [4,6]. 67 Rice blast disease is one of the most devastating diseases threatening rice production worldwide  [11][12][13][14]. They fulfill diverse roles, including suppression of chitin-triggered immunity [13][14]. By 75 contrast, cytoplasmic effectors are secreted via a plant membrane-derived biotrophic interfacial 76 complex (BIC), using a Golgi-independent process [8,[11][12]. These effectors enable M. oryzae to 77 grow in epidermal tissue and move from cell to cell using pit field sites [15]. A fungal nitronate 5 78 monooxygenase, Nmo2, involved in the nitrooxidative stress response, is also required to avoid 79 triggering plant immunity, thereby facilitating growth and BIC development of M. oryzae [16]. Host 80 cells begin to lose viability, once invasive hyphae begin to invade adjacent cells and the switch to 81 necrotrophic growth. This accompanies the appearance of necrotic disease lesions, from which the 82 fungus sporulates [10]. 83 In this study, we set out to explore the mechanism by which M. oryzae switches from biotrophic 84 to necrotrophic growth. Specifically, we decided to test the hypothesis that modulation of host 85 cellular pH may be involved in the regulation of this morphogenetic and physiological switch [17]. 86 Alkalinization of plant cells is an important early immune response to attack by pathogens [18][19][20][21], 87 and some pathogens have developed mechanisms to alter pH of plant tissues [17,22]. The 88 necrotrophic pathogens Athelia rolfsii and Sclerotinia sclerotiorum both, for instance, generate 89 oxalic acid, leading to a sharp drop in the pH of host cells [23][24], while Fusarium oxysporum 90 secretes a rapid alkalinization factor to induce host tissue alkalinization [25]. 91 Fungi are generally more acidophilic and have evolved array of mechanisms to adapt to ambient 92 alkaline pH, including the well-known PacC signaling pathway [ repressors is still relatively poorly understood. 129 Here, we show that upon infection by M. oryzae, host plant cells are alkalinized to pH 7.8 during 130 the biotrophic growth stage, but then acidified to pH 6.5 at the onset of necrotrophic growth. We 131 report that fungal adaptation to host alkalinization and the induction of host acidification requires the 132 PacC pH signaling pathway. We used a forward genetic screen to identify mutants that were 133 sensitive to alkaline pH, and found that they were all impaired in virulence. We went on to 134 characterise the PacC signaling pathway in the rice blast fungus. We show that in M. oryzae the 135 PacC transcription factor simultaneously exists as both a truncated transcriptional activator and a 136 full-length transcriptional repressor, that both localize to the nucleus during biotrophic growth of M. 137 oryzae. PacC acts as a key transcriptional regulator which coordinates expression of more than 25% 138 of the protein-encoding genes in M. oryzae to facilitate the hemibiotrophic switch, which is 139 necessary for rice blast disease. Plant cellular pH is therefore likely to be a key regulatory signal, that 140 is perceived and modulated by M. oryzae to control hemibiotrophic growth.   Table). To identify the genes disrupted in these nine  Table). In addition, these mutants 187 were reduced in conidiation by 80-90%; compared to the isogenic wild type strain ( Fig 2B). In  To our surprise, these mutants displayed similar phenotypes to the wild type, including pH sensitivity 198 conidiation and virulence (Fig 2A-2C). Therefore, PalA, PalI and Vps32 are dispensable for 199 regulating the alkaline pH response and virulence, suggesting that these three genes are not required However, ΔpalI, ΔpalA and Δvps32 mutants were similar to the wild type P131 in development of 213 IH. These data indicate that M. oryzae has a PacC pathway that is crucial for biotrophic growth. 214 To investigate whether subsequent stages of infection are affected by loss of PacC signaling, 215 we inoculated wounded rice leaves to circumvent the need for appressorium-mediated penetration. 216 The wild type P131 generated large lesions, while PacC pathway mutants, including ΔpacC, formed 217 significantly smaller lesions without evident necrosis ( Fig 2F). We stained ΔpacC-infected barley 218 leaves with Trypan blue, and observed that the ΔpacC mutant led to host cell death at a much 219 delayed time (Fig 1E), suggesting that the mutant is deficient both in its ability to undertake 220 biotrophic growth and its switch to necrotrophy. In addition, the ΔpacC mutant induced less reactive 221 oxygen species (ROS) generation in host cells. However, inhibition of ROS production by 222 diphenyleneiodonium (DPI) treatment failed to allow IH to recover growth (Fig 2G), suggesting that 223 the reduced IH growth of PacC pathway mutants is due to factors other than ROS production. 224 We also monitored pH changes in barley cells infected by the ΔpacC mutant and observed that 225 pH in the initial host cells became alkalinized at 12 hpi，and then peaked at 7.8 at 24 hpi for the 226 initially colonized plant cells and at 36 hpi for the secondary infected cells, respectively ( Fig 1B). 227 However, acidification of host cells infected by the ΔpacC mutant was much delayed (Fig 1B-1C).

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These results indicate that the PacC pathway is necessary for inducing host cellular acidification but 229 not for host cellular alkalinization. ΔpacC mutant (Fig 5B). Subsequent phenotypic assays showed that all the resulting transformants 236 were recovered in colony growth, conidiation and virulence ( Fig 5G-5H), indicating that the 237 GFP-fused PacC is functional. Interestingly, GFP signals were predominantly localized to the 238 nucleus in primary and branched IH from 18 to 30 hpi, but were then mainly distributed in the 239 cytoplasm from 36 hpi onwards ( Fig 3A). Furthermore, GFP signals reappeared in the nuclei of IH 240 that had penetrated neighboring cells between 36 and 42 hpi, and then disappeared again from the 241 nucleus at 48 hpi ( Fig 3A). By contrast, in pre-penetration stage structures, conidia and appressoria, 242 GFP signals were evenly distributed in the cytoplasm ( Fig 3A). These data reveal that PacC localizes 243 to the nucleus specifically during biotrophic invasive growth. 244 Because host cells are alkalinized and acidified during biotrophic and necrotrophic growth, 245 respectively, we suspected that host cellular pH was the inductive signal for PacC nuclear We also introduced the eGFP-PacC construct independently into the ΔpalH, ΔpalF, ΔpalC and 254 ΔpalB mutants, and examined GFP subcellular localization. GFP-PacC was evenly distributed in the 255 cytoplasm, but not in the nucleus of ΔpalH, ΔpalF, ΔpalC and ΔpalB transformants (Fig 3D). 256 Therefore, the alkaline pH-induced nuclear localization of PacC requires PalB, PalC, PalF and 257 PalH.  kDa protein, and a 36 kDa protein were detected together with a protein > 52kDa. These data 308 suggested that the 36 kDa and 52 kDa proteins may be generated from the full length eGFP-PacC 559 309 by processing, probably at the 80th and 222th aa sites (Fig 5C), respectively. PacC-binding GCCAAG consensus in their promoters were randomly selected for qRT-PCR analysis 323 with total RNA from the WT, the ΔpacC, NGP245, and NGP559 strains. Based on their expression 324 patterns, these genes were classified into three types (Fig 6). There were six type I genes, which were 325 repressed under alkaline conditions in the WT and NGP559, but up-regulated in the ΔpacC mutant 326 and NGP245 (Fig 6A), indicating that they are repressed by PacC 559 . Fourteen type II genes were 327 up-regulated in the wild type, NGP245 and NGP559 strains, but significantly reduced in the ΔpacC mutant (Fig 6B), confirming that PacC 222 is a transcription activator. We further assayed the 329 phenotypes of NGP96, NGP245 and NGP559 strains. Sensitivity to alkaline pH, conidiation and 330 virulence were fully complemented in NGP559, but only partially so in NGP245. No obvious 331 difference was observed between the ΔpacC mutant and NGP96 (Fig 5F-5H

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MGG_01779 is a PacC-regulated Type II gene probably encoding a novel C6 zinc DNA binding 342 domain protein (Fig 7), named PAG1 (for PacC Activated Gene 1). Its promoter has two GCCAAG 343 sites bound by PacC (Fig 7B). The Δpag1 mutant showed no obvious defects in colony growth or 344 conidiation (Fig 7C and D, S4J Fig), but was significantly impaired in IH branching and in virulence 345 ( Fig 7E). PAG1 therefore functions downstream of PacC to regulate biotrophic growth of IH.  (Fig 2A). 358 Taken together, these results indicate that PacC may orchestrate distinct developmental 359 processes in M. oryzae, by directly regulating a wide range of transcription factors, thereby leading 360 to large-scale transcriptional changes which are essential for establishing blast disease.

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Many of the most important plant pathogenic fungi are hemibiotrophs which are able to switch from 363 biotrophic growth and necrotrophic growth during plant infection [1-3,9-10,]. Very little, however, is 364 known regarding how these fungi switch between the two growth habits and, indeed, which signals 365 from the plant elicit such a dramatic developmental and physiological change. In this study, we 366 identified host cellular pH as an inductive signal for the necrotrophic switch in M. oryzae. 367 We found that that host cells around infection sites are initially and temporally alkalinized to pH 368 7.8 during biotrophic growth, but acidification to pH 6.5 then follows during necrotrophic growth 369 ( Figure 1A-1B and 1D-1E). This observation contrasts with a previous study which indicated that 370 host cells infected by M. oryzae remains alkaline for up to 60 hpi [51]. Our results also showed that 371 pH alkalinization is independent of PacC function (Fig 1B-1C and 1E) and is clearly inhibitory to 372 fungal growth and conidiation, based on in vitro studies (Fig 1F-1G . Moreover, acidified pH is conducive to fungal growth and conidiation in vitro (Fig 1F-1G). 377 Therefore, biotrophic growth may require a mechanism of fungal adaptation to host pH alkalinization 378 while necrotrophic growth is an active process by which the fungus prepares for future propagation. 379 Many previous studies have showed that the PacC transcription factor is important for virulence 380 of plant fungal pathogens [54][55][56][57][58], but our study, however, offers a potential mechanism which 381 indicates that the PacC pathway (including the PacC transcription factor) is involved in the 382 regulation of fungal hemibiotrophy. We have shown that the PacC pathway is required for M. oryzae 383 not only to adapt to the host cellular alkalinized pH for biotrophic growth, but also to induce pH 384 acidification, which allows necrotrophic growth (Fig 1; Fig 2). In particular, the regulation of 385 hemibiotrophy involves shuttling of the PacC transcription factor between the nucleus and the 18 386 cytoplasm, whereby PacC localizes to the nucleus during biotrophic growth and alkaline pH, but to 387 the cytoplasm during necrotrophic growth and acidic conditions (Fig 2; Fig 5A). Interestingly, palH 388 in A. nidulans mechanistically resembles mammalian GPCRs [59], and this study has revealed its 389 requirement for virulence of M. oryzae ( Fig. 2A-2E). When considered together, PalH there may be a which are required for its biological function (Fig 5 and Fig 6). To achieve their distinct 400 transcriptional functions, M. oryzae PacC forms translocate to the nucleus during biotrophic growth 401 (Fig 3A), likely as a consequence of alkaline pH (Fig 1A-1B; Fig 3B) Our results provide evidence that M. oryzae PacC activates genes that are specifically expressed 424 during biotrophic growth, and at the same time repress expression of genes that are related to 425 saprophytic growth (Fig.4A-4C). Over 2700 genes are differentially expressed in biotrophic IH of M. 426 oryzae when compared with a ΔpacC mutant ( Fig. 4A; S1 Dataset). Therefore, more than 25% of the 427 total protein-encoding genes in M. oryzae genome are regulated by PacC. This is much higher than 428 the number regulated by PacC in other plant pathogenic fungi [54,56], where mycelium grown in 429 axenic conditions was used for RNA-Seq analysis. This is, however, the first time that biotrophic 430 infection has been analyzed for PacC regulation. We confirmed that M. oryzae PacC can bind to the 431 GCCAAG cis-element, as previously reported for A. nidulans PacC (Fig.5E, Fig.7B, S5B Fig., S6B 432 Fig., S7B Fig) [38]. By surveying PacC binding motifs in promoters of each differentially expressed 433 gene, we have identified nearly 1500 genes that are very likely to be directly regulated by PacC in M. 434 oryzae biotrophic IH (Fig 4A; S4 Dataset). It is notable that among the putatively direct targets of 435 PacC are genes enriched in those encoding extracellular and plasma membrane proteins (Fig 4D-4F; 20 436 S4 Dataset), that are likely to be involved in suppression of plant immunity, remodeling fungal cell 437 walls and acquisition of carbon and nitrogen sources from living host cells (Fig 4C-4D where the activator isoform enhances gene expression associated with biotrophic growth, while the 449 repressor isoform represses genes associated with necrotrophic growth and conidiation (Fig 8B). 450 After extensive tissue colonization, M. oryzae induces disintegration and acidification of host plant 451 cells, which induces the two PacC isoforms to translocate from the nucleus thereby de-repressing 452 gene expression associated with necrotrophic growth and conidiation (Fig 8C). The shuttling of PacC 453 between the nucleus and cytoplasm according to pH in host tissue may thereby regulate the temporal 454 switch between biotrophic and necrotrophic growth that is essential for blast disease.

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Strains and culture conditions 457 The P131 strain of M. oryzae was used for all genetic manipulation and infection assays [63]. S1528, 458 which has the opposite mating type to P131, was used only for genetic crossing and co-segregation 459 analyses, as previously described [64]. Strains 70-15 and DG-ZX-83, together with P131, were only 460 used to assay effect of pH on colony growth and conidiation. All the wild-type strains and 461 transformants (Supplemental Table 4) were maintained on oatmeal tomato agar (OTA) plates at 462 28°C, as described [63]. For assaying colony growth under normal condition, mycelial blocks (φ=5 gene, 1.5 kb upstream and 1.5 kb downstream sequences were amplified and cloned into pKNH [63]. 498 The resulting vectors were independently transformed into P131. Transformants resistant to 499 hygromycin, but sensitive to neomycin, were subjected to screening by PCR. Gene deletion 500 candidates were further confirmed by Southern blot analysis. 501 For genetic complementation, genomic DNA fragments of individual PalB, PalC, PalF, PalH, 502 and PacC genes containing 1.5 kb promoter and 0.5 kb terminator regions were amplified and cloned 503 into pKN [63]. To generate the eGFP-PacC fusion construct pKGPacC 559 , a fragment amplified with 504 primers PacCNP5 and PacCNP3 was digested with XhoI and HindIII and cloned into pKNTG [63]. 505 The same strategy was used to generate the pKGPacC 245  un-labelled DNAs were used as 10× and 100× specific competitors, respectively. The samples were 560 separated on 8% native PAGE gels for 50 min, which were exposed to X-ray film for 1 h and 561 detected by a storage phosphor system (Cyclone, PerkinElmer, USA).