The Arabidopsis Ethylene/Jasmonic Acid-NRT Signaling Module Coordinates Nitrate Reallocation and the Trade-Off between Growth and Environmental Adaptation

Stresses decouple nitrate assimilation and photosynthesis through stress-initiated nitrate allocation to roots (SINAR), which is mediated by the nitrate transporters NRT1.8 and NRT1.5 and functions to promote stress tolerance. However, how SINAR communicates with the environment remains unknown. Here, we present biochemical and genetic evidence demonstrating that in Arabidopsis thaliana, ethylene (ET) and jasmonic acid (JA) affect the crosstalk between SINAR and the environment. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that ethylene response factors (ERFs), including OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59, bind to the GCC boxes in the NRT1.8 promoter region, while ETHYLENE INSENSITIVE3 (EIN3) binds to the EIN3 binding site motifs in the NRT1.5 promoter. Genetic assays showed that cadmium and sodium stresses initiated ET/JA signaling, which converged at EIN3/EIN3-Like1 (EIL1) to modulate ERF expression and hence to upregulate NRT1.8. By contrast, ET and JA signaling mediated the downregulation of NRT1.5 via EIN3/EIL1 and other, unknown component(s). SINAR enhanced stress tolerance and decreased plant growth under nonstressed conditions through the ET/JA-NRT1.5/NRT1.8 signaling module. Interestingly, when nitrate reductase was impaired, SINAR failed to affect either stress tolerance or plant growth. These data suggest that SINAR responds to environmental conditions through the ET/JA-NRT signaling module, which further modulates stress tolerance and plant growth in a nitrate reductase-dependent manner.     

Chloroplast Activity and 3′phosphadenosine 5′phosphate Signaling Regulate Programmed Cell Death in Arabidopsis

Programmed cell death (PCD) is a crucial process both for plant development and responses to biotic and abiotic stress. There is accumulating evidence that chloroplasts may play a central role during plant PCD as for mitochondria in animal cells, but it is still unclear whether they participate in PCD onset, execution, or both. To tackle this question, we have analyzed the contribution of chloroplast function to the cell death phenotype of the myoinositol phosphate synthase1 (mips1) mutant that forms spontaneous lesions in a light-dependent manner. We show that photosynthetically active chloroplasts are required for PCD to occur in mips1, but this process is independent of the redox state of the chloroplast. Systematic genetic analyses with retrograde signaling mutants reveal that 3′-phosphoadenosine 5′-phosphate, a chloroplast retrograde signal that modulates nuclear gene expression in response to stress, can inhibit cell death and compromises plant innate immunity via inhibition of the RNA-processing 5′-3′ exoribonucleases. Our results provide evidence for the role of chloroplast-derived signal and RNA metabolism in the control of cell death and biotic stress response.Programmed cell death (PCD) is a universal process in multicellular organisms, contributing to the controlled and active degradation of the cell. In plants, PCD is required for processes as diverse as development, self-incompatibility, and stress response. One well-documented example is the induction of PCD upon pathogen attack, allowing the confinement of the infection, and resistance of the plant. The signaling events leading to the onset of PCD have been extensively studied: pathogen recognition triggers activation of mitogen-activated protein kinase cascades, as well as production of reactive oxygen species (ROS) and salicylic acid (SA), which lead to a hypersensitive response (Coll et al., 2011).From a cellular point of view, several classes of plant PCD have been described and compared with the ones found in animal cells (van Doorn, 2011). PCD is thought to have evolved independently in plants and animals, and genes underlying these mechanisms are therefore poorly conserved between the two kingdoms. However, most cellular features are conserved between plant and animal PCD that are both characterized by cell shrinkage, chromatin condensation, DNA laddering, mitochondria permeabilization, and depolarization (Dickman and Fluhr, 2013). In animal cells, mitochondria play a central role in the regulation of apoptosis (Czabotar et al., 2014; Mariño et al., 2014), and this role is likely shared between the two kingdoms (Lord and Gunawardena, 2012). That said, additional mitochondria-independent PCD pathways have clearly evolved in plants.Genetic approaches have greatly contributed to our understanding of cellular pathways governing PCD in plants. For example, the isolation of lesion mimic mutants (LMMs), in which cell death occurs spontaneously, has allowed the identification of several negative regulators of cell death (for review, see Bruggeman et al., 2015b). Interestingly, lesion formation is light dependent in several of these mutants, which include one of the best characterized LMMs—lesions simulating disease1 (lsd1; Dietrich et al., 1994). The LSD1 protein is required for plant acclimation to excess excitation energy (Mateo et al., 2004): when plants are exposed to excessive amounts of light, the redox status of the plastoquinone pool in the chloroplastic electron transfer chain is thought to influence LSD1-dependent signaling to modulate cell death (Mühlenbock et al., 2008). Additionally, we have previously identified the myoinositol phosphate synthase1 (mips1) mutant as a LMM, in which lesion formation is also light dependent (Meng et al., 2009). This mutant is deficient in the myoinositol (MI) phosphate synthase that catalyzes the first committed step of MI biosynthesis and displays pleiotropic defects such as reduced root growth, abnormal vein development, and spontaneous cell death on leaves, together with severe growth reduction after lesions begin to develop (Meng et al., 2009; Donahue et al., 2010). The light-dependent PCD in the mips1 mutant, as observed for lsd1, suggests that chloroplasts may play a role in the MI-dependent cell death regulation. Accumulating evidence suggests that chloroplasts may play a central role in PCD regulation like mitochondria in animal cells (Wang and Bayles, 2013). First, as described in the case of lsd1, excess light energy received by the chloroplast can function as a trigger for PCD. Furthermore, singlet oxygen (¹O₂), a ROS, can activate the EXECUTER1 (EX1) and EX2 proteins in the chloroplasts to initiate PCD (Lee et al., 2007). Likewise, ROS generated by chloroplasts play a major role for PCD onset during nonhost interaction between tobacco (Nicotiana tabacum) and Xanthomonas campestris (Zurbriggen et al., 2009). Finally, functional chloroplasts have also been shown to be required for PCD in cell suspensions (Gutierrez et al., 2014) and in a number of LMMs (Mateo et al., 2004; Meng et al., 2009; Bruggeman et al., 2015b). Thus, chloroplasts are now recognized as important components of plant defense response against pathogens (Stael et al., 2015) and are proposed to function with mitochondria in the execution of PCD (Van Aken and Van Breusegem, 2015). However, the exact signaling and metabolic contribution of chloroplasts to PCD remain to be elucidated. Furthermore, cross talk between chloroplasts and mitochondria does occur, such as during photorespiration (Sunil et al., 2013), but whether such communication functions sequentially or in parallel in the control of PCD remains to be determined (Van Aken and Van Breusegem, 2015).To further investigate how chloroplasts contribute to the regulation of cell death, we performed both forward and reverse genetics on the mips1 mutant. An extragenic secondary mutation in divinyl protochlorophyllide 8-vinyl reductase involved in chlorophyll biosynthesis leads to chlorophyll deficiency that abolishes the mips1 cell death phenotype, as do changes in CO₂ availability. These findings provide evidence for a link between photosynthetic activity and PCD induction in mips1. Additionally, we investigated the contribution of several retrograde signaling pathways (Chan et al., 2015) to the control of PCD in mips1. This process was independent of GENOMES UNCOUPLED (GUN) and EX signaling pathways, but we found that the SAL1-PAP_XRN retrograde signaling pathway inhibits cell death as well as basal defense reactions in Arabidopsis (Arabidopsis thaliana).     

Probing Arabidopsis Chloroplast Diacylglycerol Pools by Selectively Targeting Bacterial Diacylglycerol Kinase to Suborganellar Membranes

Diacylglycerol (DAG) is an intermediate in metabolism of both triacylglycerols and membrane lipids. Probing the steady-state pools of DAG and understanding how they contribute to the synthesis of different lipids is important when designing plants with altered lipid metabolism. However, traditional methods of assaying DAG pools are difficult, because its abundance is low and because fractionation of subcellular membranes affects DAG pools. To manipulate and probe DAG pools in an in vivo context, we generated multiple stable transgenic lines of Arabidopsis (Arabidopsis thaliana) that target an Escherichia coli DAG kinase (DAGK) to each leaflet of each chloroplast envelope membrane. E. coli DAGK is small, self inserts into membranes, and has catalytic activity on only one side of each membrane. By comparing whole-tissue lipid profiles between our lines, we show that each line has an individual pattern of DAG, phosphatidic acid, phosphatidylcholine, and triacylglycerol steady-state levels, which supports an individual function of DAG in each membrane leaflet. Furthermore, conversion of DAG in the leaflets facing the chloroplast intermembrane space by DAGK impairs plant growth. As a result of DAGK presence in the outer leaflet of the outer envelope membrane, phosphatidic acid accumulation is not observed, likely because it is either converted into other lipids or removed to other membranes. Finally, we use the outer envelope-targeted DAGK line as a tool to probe the accessibility of DAG generated in response to osmotic stress.Diacylglycerol (DAG) is a central metabolite in plant lipid metabolism. Its glycerol backbone is modified with two acyl chains. If a third acyl chain is added, triacylglycerol (TAG) is formed, whereas if a head group is added, it is converted into polar lipids such as a galactolipid. In green tissues, the majority of DAG is used as an intermediate in galactolipid synthesis, because the extensive thylakoid membrane system consists of approximately 85% galactolipids (Block et al., 1983). Although under normal conditions the galactolipids are exclusively chloroplastic, in Arabidopsis (Arabidopsis thaliana), the DAG used to make galactolipids is derived from assembly pathways in both the chloroplast and the endoplasmic reticulum (ER; Benning, 2009). In both pathways, the bulk of the fatty acids are synthesized in the chloroplast stroma (Browse et al., 1986) in the following order of abundance: 18:1, 16:0, and 18:0 (Wallis and Browse, 2002).In the chloroplast pathway, these fatty acids are directly attached to a glycerol-3-P, generating first lyso-phosphatidic acid (l-PtdOH) and then phosphatidic acid (PtdOH) in the inner leaflet of the chloroplast inner envelope (Fig. 1; Frentzen et al., 1983). The acyltransferases involved are specific to the extent that the sn-2 position of the glycerol backbone predominantly receives a 16:0 fatty acid. PtdOH is then used directly for phosphatidylglycerol (PtdGro) synthesis (Babiychuk et al., 2003) or converted to DAG by a PtdOH phosphatase (Joyard and Douce, 1977). The PtdOH phosphatase activity is known to be associated with the inner envelope, though which leaflet is obscured by the fact that DAG can efficiently flip across membranes (Hamilton et al., 1991) and the actual enzyme has not been unambiguously identified and located (Nakamura et al., 2007). However, the leaflet associations of two other enzymes that use DAG in the inner envelope have been established. MGD1, which uses DAG to synthesize the most abundant galactolipid, monogalactosyldiacylglycerol (MGDG), is on the outer leaflet of the inner envelope membrane (Xu et al., 2005), while SQD2, which uses DAG to generate the less abundant sulfolipid, sulfoquinovosyldiacylglycerol (SQDG), is located on the inner leaflet of the inner envelope membrane (Tietje and Heinz, 1998). Also associated with the inner envelope membrane are a number of fatty acid desaturases, including FAD4, FAD5, FAD6, FAD7, and FAD8 (Joyard et al., 2010). Two of these are specific, generating lipids with signature desaturations: FAD4 desaturates only the 16:0 fatty acid of PtdGro, giving plastidic PtdGro a distinct 16:1 Δ3 trans moiety (Browse et al., 1985; Gao et al., 2009), and FAD5 desaturates primarily the 16:0 fatty acid of MGDG, producing 16:1 Δ7 cis (Kunst et al., 1989). The remaining desaturases are less specific, with little preference for head group or acyl tail. They further desaturate 16:1 or 18:1 in the cis conformation to 16:2 or 18:2 (FAD6; Browse et al., 1989) and on to 16:3 or 18:3 (FAD7 and FAD8; Wallis and Browse, 2002). The combined actions of these FADs result in the highly desaturated fatty acid profiles seen for most chloroplast lipids.Open in a separate windowFigure 1.Overview of DAG pools in the chloroplast envelope membranes. Processes that are known to have activity feeding into or withdrawing from DAG pools in the chloroplast envelope membranes are shown. Enzymes are indicated, and their substrates and products are connected with black arrows. However, for space reasons, not all reactants are shown. Membrane leaflets are indicated, and enzymes with known membrane topology are displayed correctly, while those without known topology are displayed in the center of the appropriate membrane. The acyl group preferred by each l-PtdOH acyltransferase is given in parentheses. Proposed processes transporting lipids from the ER to the chloroplast are shown with dashed arrows. Enzymes are as follows: 1, ATS1; 2, ATS2; 3, lipid phosphate phosphatase γ; 4, MGD1; 5, SQD2; 6, cytosolic phospholipases; 7, MGD2 or MGD3; 8, SFR2; 9, acyl-CoA:glycerol-3-P acyltransferase; 10, l-PtdOH acyltransferase; 11, PtdOH phosphatase; 12, cytidine diphosphate-choline:DAG cholinephosphotransferase; 13, TGD4; and 14, TGD1, TGD 2, TGD3 lipid transport complex. OE, Chloroplast outer envelope membrane; IE, chloroplast inner envelope membrane; ACP, acyl carrier protein. [See online article for color version of this figure.]In unstressed plants, DAG seems to be used primarily in the inner chloroplast envelope. However, several conditions are known to cause extensive DAG use in the chloroplast outer envelope. During phosphate deprivation, MGD2 and MDG3 synthesize MGDG from DAG on the outer leaflet of the outer envelope membrane (Kobayashi et al., 2009). The DAG backbones are probably supplied from the phosphatidylcholine (PtdCho) pool by phospholipase activity, which was shown to be simultaneously up-regulated (Andersson et al., 2004; Nakamura et al., 2005). DAG is also generated during freezing stress by a galactolipid:galactolipid galactosyltransferase named Sensitive to FReezing2 (SFR2). This enzyme transfers the galactosyl head group of MGDG onto another MGDG, giving rise to digalactosyldiacylglycerol (DGDG) and DAG (Moellering et al., 2010). The DAG is subsequently sequestered into a lipid droplet by formation of TAG by an as yet unidentified enzyme.In the ER pathway, fatty acids synthesized in the chloroplast stroma are exported through a still poorly defined mechanism to the ER and activated to acyl-CoAs. Acyltransferases sequentially catalyze formation of l-PtdOH and PtdOH from glycerol-3-P and acyl-CoAs. Again, the acyltransferase working on the sn-2 position of the glycerol backbone is specific, but unlike the chloroplast isoform, it prefers an 18:1 carbon fatty acid (Frentzen et al., 1983). Newly generated PtdOH can be converted to PtdGro or phosphatidyl inositol (PtdIns) (Collin et al., 1999) or hydrolyzed to DAG (Shimojima et al., 2009). DAG can then be further metabolized to TAG and PtdCho. PtdCho acyl groups (18:1/18:1 and 18:1/16:0) are desaturated sequentially by desaturases FAD2 (Okuley et al., 1994) and FAD3 (Browse et al., 1993). These desaturases prefer PtdCho as substrate. The acyl chains modified on PtdCho are transferred to other ER lipids, including DAG, as a result of continual acyl editing of the PtdCho pool (Bates et al., 2012). Furthermore, PtdOH and many of the other extraplastidic phospholipids can be converted to DAG by action of phospholipases (Shimojima et al., 2009). These have as yet partially defined roles in response to stress or recycling of membrane lipids (Testerink and Munnik, 2005).Glycerolipid precursors generated by de novo synthesis, acyl editing, and possibly stress conditions in the ER are transported to the chloroplast by a mechanism that is likely to involve at least two putative lipid transporters: trigalactosyldiacylglycerol4 (TGD4) in the chloroplast outer envelope membrane and the TGD1, TGD2, and TGD3 complex in the inner envelope membrane (Wang and Benning, 2012). The actual lipid species transported remains unclear, but PtdCho, lyso-phosphatidylcholine, PtdOH, and DAG have been discussed in the literature (Andersson and Dörmann, 2009). The DAG moieties are then fully incorporated into all plastidic lipids except PtdGro, presumably using the same pathways that metabolize plastidic DAG, described above. Because of the preference of chloroplast and ER sn-2 acyltransferases for 16 or 18 carbon fatty acids, respectively, the origin of the DAG moieties can be distinguished by positional analysis of the acyl groups on the glycerol backbone (Roughan and Slack, 1982). In Arabidopsis, the chloroplast and ER lipid synthesis pathways contribute nearly equally to mature chloroplast lipids (Browse et al., 1986; Mongrand et al., 1998). Thus, the DAG pools described so far in the chloroplast inner and outer envelope membranes are each of dual origin.A challenge for the analysis of the different DAG pools is that this compound is not a bilayer-forming lipid and thus does not accumulate stably to high levels. Furthermore, during any lengthy fractionation procedure, its levels can be expected to alter, as DAG-modifying enzymes exist in multiple membranes. Moreover, because DAG is quickly metabolized and may have efficient transport systems (Dong et al., 2012), it is difficult to confirm whether metabolizing enzymes are accessing the same or separate DAG pools.To probe different DAG pools of chloroplast membranes in vivo, we have generated a series of stable transgenic Arabidopsis lines in which we target an Escherichia coli DAG kinase (DAGK) to each leaflet of the chloroplast envelope membranes. The basic utility of this approach was previously shown by targeting a DAGK to the chloroplast in tobacco (Nicotiana tabacum) using a single construct fusing the bacterial protein to the Rubisco small subunit N-terminal peptide (Fritz et al., 2007). Here, we present a full phenotypic analysis of these lines, determining which chloroplast membranes show steady-state alterations of DAG and PtdOH levels predicted by ectopic DAGK activity. We further determine the accessibility of DAG pools generated on the outer leaflet of the chloroplast outer envelope membrane during osmotic stress. Having this system established in Arabidopsis will allow characterization of DAG pools in multiple lipid mutant lines.     

HYPERSENSITIVE TO HIGH LIGHT1 Interacts with LOW QUANTUM YIELD OF PHOTOSYSTEM II1 and Functions in Protection of Photosystem II from Photodamage in Arabidopsis

Under high-irradiance conditions, plants must efficiently protect photosystem II (PSII) from damage. In this study, we demonstrate that the chloroplast protein HYPERSENSITIVE TO HIGH LIGHT1 (HHL1) is expressed in response to high light and functions in protecting PSII against photodamage. Arabidopsis thaliana hhl1 mutants show hypersensitivity to high light, drastically decreased PSII photosynthetic activity, higher nonphotochemical quenching activity, a faster xanthophyll cycle, and increased accumulation of reactive oxygen species following high-light exposure. Moreover, HHL1 deficiency accelerated the degradation of PSII core subunits under high light, decreasing the accumulation of PSII core subunits and PSII–light-harvesting complex II supercomplex. HHL1 primarily localizes in the stroma-exposed thylakoid membranes and associates with the PSII core monomer complex through direct interaction with PSII core proteins CP43 and CP47. Interestingly, HHL1 also directly interacts, in vivo and in vitro, with LOW QUANTUM YIELD OF PHOTOSYSTEM II1 (LQY1), which functions in the repair and reassembly of PSII. Furthermore, the hhl1 lqy1 double mutants show increased photosensitivity compared with single mutants. Taken together, these results suggest that HHL1 forms a complex with LQY1 and participates in photodamage repair of PSII under high light.     

Arabidopsis DELLA and JAZ Proteins Bind the WD-Repeat/bHLH/MYB Complex to Modulate Gibberellin and Jasmonate Signaling Synergy

Integration of diverse environmental and endogenous signals to coordinately regulate growth, development, and defense is essential for plants to survive in their natural habitat. The hormonal signals gibberellin (GA) and jasmonate (JA) antagonistically and synergistically regulate diverse aspects of plant growth, development, and defense. GA and JA synergistically induce initiation of trichomes, which assist seed dispersal and act as barriers to protect plants against insect attack, pathogen infection, excessive water loss, and UV irradiation. However, the molecular mechanism underlying such synergism between GA and JA signaling remains unclear. In this study, we revealed a mechanism for GA and JA signaling synergy and identified a signaling complex of the GA pathway in regulation of trichome initiation. Molecular, biochemical, and genetic evidence showed that the WD-repeat/bHLH/MYB complex acts as a direct target of DELLAs in the GA pathway and that both DELLAs and JAZs interacted with the WD-repeat/bHLH/MYB complex to mediate synergism between GA and JA signaling in regulating trichome development. GA and JA induce degradation of DELLAs and JASMONATE ZIM-domain proteins to coordinately activate the WD-repeat/bHLH/MYB complex and synergistically and mutually dependently induce trichome initiation. This study provides deep insights into the molecular mechanisms for integration of different hormonal signals to synergistically regulate plant development.     

Degradation of the Endoplasmic Reticulum by Autophagy during Endoplasmic Reticulum Stress in Arabidopsis

In this article, we show that the endoplasmic reticulum (ER) in Arabidopsis thaliana undergoes morphological changes in structure during ER stress that can be attributed to autophagy. ER stress agents trigger autophagy as demonstrated by increased production of autophagosomes. In response to ER stress, a soluble ER marker localizes to autophagosomes and accumulates in the vacuole upon inhibition of vacuolar proteases. Membrane lamellae decorated with ribosomes were observed inside autophagic bodies, demonstrating that portions of the ER are delivered to the vacuole by autophagy during ER stress. In addition, an ER stress sensor, INOSITOL-REQUIRING ENZYME-1b (IRE1b), was found to be required for ER stress–induced autophagy. However, the IRE1b splicing target, bZIP60, did not seem to be involved, suggesting the existence of an undiscovered signaling pathway to regulate ER stress–induced autophagy in plants. Together, these results suggest that autophagy serves as a pathway for the turnover of ER membrane and its contents in response to ER stress in plants.     

Cell Growth Defect Factor1/CHAPERONE-LIKE PROTEIN OF POR1 Plays a Role in Stabilization of Light-Dependent Protochlorophyllide Oxidoreductase in Nicotiana benthamiana and Arabidopsis

Angiosperms require light for chlorophyll biosynthesis because one reaction in the pathway, the reduction of protochlorophyllide (Pchlide) to chlorophyllide, is catalyzed by the light-dependent protochlorophyllide oxidoreductase (POR). Here, we report that Cell growth defect factor1 (Cdf1), renamed here as CHAPERONE-LIKE PROTEIN OF POR1 (CPP1), an essential protein for chloroplast development, plays a role in the regulation of POR stability and function. Cdf1/CPP1 contains a J-like domain and three transmembrane domains, is localized in the thylakoid and envelope membranes, and interacts with POR isoforms in chloroplasts. CPP1 can stabilize POR proteins with its holdase chaperone activity. CPP1 deficiency results in diminished POR protein accumulation and defective chlorophyll synthesis, leading to photobleaching and growth inhibition of plants under light conditions. CPP1 depletion also causes reduced POR accumulation in etioplasts of dark-grown plants and as a result impairs the formation of prolamellar bodies, which subsequently affects chloroplast biogenesis upon illumination. Furthermore, in cyanobacteria, the CPP1 homolog critically regulates POR accumulation and chlorophyll synthesis under high-light conditions, in which the dark-operative Pchlide oxidoreductase is repressed by its oxygen sensitivity. These findings and the ubiquitous presence of CPP1 in oxygenic photosynthetic organisms suggest the conserved nature of CPP1 function in the regulation of POR.     

Manipulation of Mitogen-Activated Protein Kinase Kinase Signaling in the Arabidopsis Stomatal Lineage Reveals Motifs That Contribute to Protein Localization and Signaling Specificity

When multiple mitogen-activated protein kinase (MAPK) components are recruited recurrently to transduce signals of different origins, and often opposing outcomes, mechanisms to enforce signaling specificity are of utmost importance. These mechanisms are largely uncharacterized in plant MAPK signaling networks. The Arabidopsis thaliana stomatal lineage was previously used to show that when rendered constitutively active, four MAPK kinases (MKKs), MKK4/5/7/9, are capable of perturbing stomatal development and that these kinases comprise two pairs, MKK4/5 and MKK7/9, with both overlapping and divergent functions. We characterized the contributions of specific structural domains of these four “stomatal” MKKs to MAPK signaling output and specificity both in vitro and in vivo within the three discrete cell types of the stomatal lineage. These results verify the influence of functional docking (D) domains of MKKs on MAPK signal output and identify novel regulatory functions for previously uncharacterized structures within the N termini of MKK4/5. Beyond this, we present a novel function of the D-domains of MKK7/9 in regulating the subcellular localization of these kinases. These results provide tools to broadly assess the extent to which these and additional motifs within MKKs function to regulate MAPK signal output throughout the plant.     

Crosstalk between the Unfolded Protein Response and Pathways That Regulate Pathogenic Development in Ustilago maydis

The unfolded protein response (UPR) is a conserved eukaryotic signaling pathway regulating endoplasmic reticulum (ER) homeostasis during ER stress, which results, for example, from an increased demand for protein secretion. Here, we characterize the homologs of the central UPR regulatory proteins Hac1 (for Homologous to ATF/CREB1) and Inositol Requiring Enzyme1 in the plant pathogenic fungus Ustilago maydis and demonstrate that the UPR is tightly interlinked with the b mating-type-dependent signaling pathway that regulates pathogenic development. Exact timing of UPR is required for virulence, since premature activation interferes with the b-dependent switch from budding to filamentous growth. In addition, we found crosstalk between UPR and the b target Clampless1 (Clp1), which is essential for cell cycle release and proliferation in planta. The unusual C-terminal extension of the U. maydis Hac1 homolog, Cib1 (for Clp1 interacting bZIP1), mediates direct interaction with Clp1. The interaction between Clp1 and Cib1 promotes stabilization of Clp1, resulting in enhanced ER stress tolerance that prevents deleterious UPR hyperactivation. Thus, the interaction between Cib1 and Clp1 constitutes a checkpoint to time developmental progression and increased secretion of effector proteins at the onset of biotrophic development. Crosstalk between UPR and the b mating-type regulated developmental program adapts ER homeostasis to the changing demands during biotrophy.