Phytophthora effector PSR1 hijacks the host pre-mRNA splicing machinery to modulate small RNA biogenesis and plant immunity.

Phytophthora effector PSR1 suppresses small RNA (sRNA)-mediated immunity in plants, but the underlying mechanism remains unknown. Here, we show that Phytophthora suppressor of RNA silencing 1 (PSR1) contributes to the pathogenicity of Phytophthora sojae and specifically binds to three conserved C-terminal domains of the eukaryotic PSR1-Interacting Protein 1 (PINP1). PINP1 encodes PRP16, a core pre-mRNA splicing factor that unwinds RNA duplexes and binds to primary microRNA transcripts and general RNAs. Intriguingly, PSR1 decreased both RNA helicase and RNA-binding activity of PINP1, thereby dampening sRNA biogenesis and RNA metabolism. The PSR1-PINP1 interaction caused global changes in alternative splicing (AS). A total of 5,135 genes simultaneously exhibited mis-splicing in both PSR1-overexpressing and PINP1-silenced plants. AS upregulated many mRNA transcripts that had their introns retained. The high occurrence of intron retention in AS-induced transcripts significantly promoted Phytophthora pathogen infection in Nicotiana benthamiana, and this might be caused by the production of truncated proteins. Taken together, our findings reveal a key role for PINP1 in regulating sRNA biogenesis and plant immunity.

An E3 Ligase Affects the NLR Receptor Stability and Immunity to Powdery Mildew.

Following the detection of pathogen cognate effectors, plant Nod-like receptors (NLRs) trigger isolate-specific immunity that is generally associated with cell death. The regulation of NLR stability is important to ensure effective immunity. In barley (Hordeum vulgare), the allelic Mildew locus A (MLA) receptors mediate isolate-specific disease resistance against powdery mildew fungus (Blumeria graminis f. sp. hordei). Currently, how MLA stability is controlled remains unknown. Here, we identified an MLA-interacting RING-type E3 ligase, MIR1, that interacts with several MLAs. We showed that the carboxyl-terminal TPR domain of MIR1 mediates the interaction with the coiled-coil domain-containing region of functional MLAs, such as MLA1, MLA6, and MLA10, but not with that of the nonfunctional MLA18-1. MIR1 can ubiquitinate the amino-terminal region of MLAs in vitro and promotes the proteasomal degradation of MLAs in vitro and in planta. Both proteasome inhibitor treatment and virus-induced gene silencing-mediated MIR1 silencing significantly increased MLA abundance in barley transgenic lines. Furthermore, overexpression of MIR1 specifically compromised MLA-mediated disease resistance in barley, while coexpression of MIR1 and MLA10 attenuated MLA10-induced cell death signaling in Nicotiana benthamiana Together, our data reveal a mechanism for the control of the stability of MLA immune receptors and for the attenuation of MLA-triggered defense signaling by a RING-type E3 ligase via the ubiquitin proteasome system.

The miR9863 family regulates distinct Mla alleles in barley to attenuate NLR receptor-triggered disease resistance and cell-death signaling.

Barley (Hordeum vulgare L.) Mla alleles encode coiled-coil (CC), nucleotide binding, leucine-rich repeat (NB-LRR) receptors that trigger isolate-specific immune responses against the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). How Mla or NB-LRR genes in grass species are regulated at post-transcriptional level is not clear. The microRNA family, miR9863, comprises four members that differentially regulate distinct Mla alleles in barley. We show that miR9863 members guide the cleavage of Mla1 transcripts in barley, and block or reduce the accumulation of MLA1 protein in the heterologous Nicotiana benthamiana expression system. Regulation specificity is determined by variation in a unique single-nucleotide-polymorphism (SNP) in mature miR9863 family members and two SNPs in the Mla miR9863-binding site that separates these alleles into three groups. Further, we demonstrate that 22-nt miR9863s trigger the biogenesis of 21-nt phased siRNAs (phasiRNAs) and together these sRNAs form a feed-forward regulation network for repressing the expression of group I Mla alleles. Overexpression of miR9863 members specifically attenuates MLA1, but not MLA10-triggered disease resistance and cell-death signaling. We propose a key role of the miR9863 family in dampening immune response signaling triggered by a group of MLA immune receptors in barley.

Barley MLA immune receptors directly interfere with antagonistically acting transcription factors to initiate disease resistance signaling.

The nucleotide binding domain and Leucine-rich repeat (NLR)-containing proteins in plants and animals mediate pathogen sensing inside host cells and mount innate immune responses against microbial pathogens. The barley (Hordeum vulgare) mildew A (MLA) locus encodes coiled-coil (CC)-type NLRs mediating disease resistance against the powdery mildew pathogen Blumeria graminis. Here, we report direct interactions between MLA and two antagonistically acting transcription factors, MYB6 and WRKY1. The N-terminal CC signaling domain of MLA interacts with MYB6 to stimulate its DNA binding activity. MYB6 functions as a positive regulator of basal and MLA-mediated immunity responses to B. graminis. MYB6 DNA binding is antagonized by direct association with WRKY1 repressor, which in turn also interacts with the MLA CC domain. The activated form of full-length MLA10 receptor is needed to release MYB6 activator from WRKY1 repression and to stimulate MYB6-dependent gene expression. This implies that, while sequestered by the WRKY1 repressor in the presence of the resting immune receptor, MYB6 acts as an immediate and positive postactivation signaling component of the active state of MLA during transcriptional reprogramming for innate immune responses.

Structure-function analysis of barley NLR immune receptor MLA10 reveals its cell compartment specific activity in cell death and disease resistance.

Plant intracellular immune receptors comprise a large number of multi-domain proteins resembling animal NOD-like receptors (NLRs). Plant NLRs typically recognize isolate-specific pathogen-derived effectors, encoded by avirulence (AVR) genes, and trigger defense responses often associated with localized host cell death. The barley MLA gene is polymorphic in nature and encodes NLRs of the coiled-coil (CC)-NB-LRR type that each detects a cognate isolate-specific effector of the barley powdery mildew fungus. We report the systematic analyses of MLA10 activity in disease resistance and cell death signaling in barley and Nicotiana benthamiana. MLA10 CC domain-triggered cell death is regulated by highly conserved motifs in the CC and the NB-ARC domains and by the C-terminal LRR of the receptor. Enforced MLA10 subcellular localization, by tagging with a nuclear localization sequence (NLS) or a nuclear export sequence (NES), shows that MLA10 activity in cell death signaling is suppressed in the nucleus but enhanced in the cytoplasm. By contrast, nuclear localized MLA10 is sufficient to mediate disease resistance against powdery mildew fungus. MLA10 retention in the cytoplasm was achieved through attachment of a glucocorticoid receptor hormone-binding domain (GR), by which we reinforced the role of cytoplasmic MLA10 in cell death signaling. Together with our data showing an essential and sufficient nuclear MLA10 activity in disease resistance, this suggests a bifurcation of MLA10-triggered cell death and disease resistance signaling in a compartment-dependent manner.

HvMPK4 phosphorylates HvWRKY1 to enhance its suppression of barley immunity to powdery mildew fungus.

Mitogen-activated protein kinase (MAPK) cascades play important roles in disease resistance in model plant species. However, the functions of MAPK signaling pathways in crop disease resistance are largely unknown. Here we report the function of HvMKK1-HvMPK4-HvWRKY1 module in barley immune system. HvMPK4 is identified to play a negative role in barley immune response against Bgh, as virus-induced gene silencing of HvMPK4 results in enhanced disease resistance whilst stably overexpressing HvMPK4 leads to super-susceptibility to Bgh infection. Furthermore, the barley MAPK kinase HvMKK1 is found to specifically interact with HvMPK4, and the activated HvMKK1DD variant specifically phosphorylates HvMPK4 in vitro. Moreover, the transcription factor HvWRKY1 is identified to be a downstream target of HvMPK4 and phosphorylated by HvMPK4 in vitro in the presence of HvMKK1DD. Phosphorylation assay coupled with mutagenesis analyses identifies S122, T284, and S347 in HvWRKY1 as the major residues phosphorylated by HvMPK4. HvWRKY1 is phosphorylated in barley at the early stages of Bgh infection, which enhances its suppression on barley immunity likely due to enhanced DNA-binding and transcriptional repression activity. Our data suggest that the HvMKK1-HvMPK4 kinase pair acts upstream of HvWRKY1 to negatively regulate barley immunity against powdery mildew.

A kinase fusion protein from Aegilops longissima confers resistance to wheat powdery mildew.

Many disease resistance genes have been introgressed into wheat from its wild relatives. However, reduced recombination within the introgressed segments hinders the cloning of the introgressed genes. Here, we have cloned the powdery mildew resistance gene Pm13, which is introgressed into wheat from Aegilops longissima, using a method that combines physical mapping with radiation-induced chromosomal aberrations and transcriptome sequencing analysis of ethyl methanesulfonate (EMS)-induced loss-of-function mutants. Pm13 encodes a kinase fusion protein, designated MLKL-K, with an N-terminal domain of mixed lineage kinase domain-like protein (MLKL_NTD domain) and a C-terminal serine/threonine kinase domain bridged by a brace. The resistance function of Pm13 is validated through transient and stable transgenic complementation assays. Transient over-expression analyses in Nicotiana benthamiana leaves and wheat protoplasts reveal that the fragment Brace-Kinase122-476 of MLKL-K is capable of inducing cell death, which is dependent on a functional kinase domain and the three α-helices in the brace region close to the N-terminus of the kinase domain.

Phytophthora sojae boosts host trehalose accumulation to acquire carbon and initiate infection.

Successful infection by pathogenic microbes requires effective acquisition of nutrients from their hosts. Root and stem rot caused by Phytophthora sojae is one of the most important diseases of soybean (Glycine max). However, the specific form and regulatory mechanisms of carbon acquired by P. sojae during infection remain unknown. In the present study, we show that P. sojae boosts trehalose biosynthesis in soybean through the virulence activity of an effector PsAvh413. PsAvh413 interacts with soybean trehalose-6-phosphate synthase 6 (GmTPS6) and increases its enzymatic activity to promote trehalose accumulation. P. sojae directly acquires trehalose from the host and exploits it as a carbon source to support primary infection and development in plant tissue. Importantly, GmTPS6 overexpression promoted P. sojae infection, whereas its knockdown inhibited the disease, suggesting that trehalose biosynthesis is a susceptibility factor that can be engineered to manage root and stem rot in soybean.

Orthologous genes Pm12 and Pm21 from two wild relatives of wheat show evolutionary conservation but divergent powdery mildew resistance.

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a devastating disease that threatens wheat production worldwide. Pm12, which originated from Aegilops speltoides, a wild relative of wheat, confers strong resistance to powdery mildew and therefore has potential use in wheat breeding. Using susceptible mutants induced by gamma irradiation, we physically mapped and isolated Pm12 and showed it to be orthologous to Pm21 from Dasypyrum villosum, also a wild relative of wheat. The resistance function of Pm12 was validated via ethyl methanesulfonate mutagenesis, virus-induced gene silencing, and stable genetic transformation. Evolutionary analysis indicates that the Pm12/Pm21 loci in wheat species are relatively conserved but dynamic. Here, we demonstrated that the two orthologous genes, Pm12 and Pm21, possess differential resistance against the same set of Bgt isolates. Overexpression of the coiled-coil domains of both PM12 and PM21 induces cell death in Nicotiana benthamiana leaves. However, their full-length forms display different cell death-inducing activities caused by their distinct intramolecular interactions. Cloning of Pm12 will facilitate its application in wheat breeding programs. This study also gives new insight into two orthologous resistance genes, Pm12 and Pm21, which show different race specificities and intramolecular interaction patterns.

Rumble in the nuclear jungle: compartmentalization, trafficking, and nuclear action of plant immune receptors.

Plants and animals have evolved structurally related innate immune sensors inside cells to detect the presence of microbial molecules. An evolutionary ancient folding machinery becomes engaged for the synthesis of autorepressed receptor forms in both kingdoms. The receptors act as regulatory signal transduction switches and are activated upon direct or indirect perception of non-self structures. Recent findings indicate that nucleo-cytoplasmic partitioning and nuclear activity is critical for the function of several plant immune sensors, thereby linking receptor function to transcriptional reprogramming of host cells for pathogen defense. This implies short signalling pathways and reveals parallels with regulatory control mechanisms of animal steroid receptors.

Molecular analysis of common wheat genes encoding three types of cytosolic heat shock protein 90 (Hsp90): functional involvement of cytosolic Hsp90s in the control of wheat seedling growth and disease resistance.

Heat shock protein 90 (Hsp90) molecular chaperones play important roles in plant growth and responses to environmental stimuli. However, little is known about the genes encoding Hsp90s in common wheat. Here, we report genetic and functional analysis of the genes specifying cytosolic Hsp90s in this species. Three groups of homoeologous genes (TaHsp90.1, TaHsp90.2 and TaHsp90.3), encoding three types of cytosolic Hsp90, were isolated. The loci containing TaHsp90.1, TaHsp90.2 and TaHsp90.3 genes were assigned to groups 2, 7 and 5 chromosomes, respectively. TaHsp90.1 genes exhibited higher transcript levels in the stamen than in the leaf, root and culm. TaHsp90.2 and TaHsp90.3 genes were more ubiquitously transcribed in the vegetative and reproductive organs examined. Decreasing the expression of TaHsp90.1 genes through virus-induced gene silencing (VIGS) caused pronounced inhibition of wheat seedling growth, whereas the suppression of TaHsp90.2 or TaHsp90.3 genes via VIGS compromised the hypersensitive resistance response of the wheat variety Suwon 11 to stripe rust fungus. Our work represents the first systematic determination of wheat genes encoding cytosolic Hsp90s, and provides useful evidence for the functional involvement of cytosolic Hsp90s in the control of seedling growth and disease resistance in common wheat.

The Powdery Mildew Effector CSEP0027 Interacts With Barley Catalase to Regulate Host Immunity.

Powdery mildew is one of the most important fungal pathogen diseases. The genome of barley mildew fungus, Blumeria graminis f. sp. hordei (Bgh), encodes a large number of candidate secreted effector proteins (CSEPs). So far, the function and mechanism of most CSEPs remain largely unknown. Here, we identify a Bgh effector CSEP0027, a member of family 41, triggering cell death in Nicotiana benthamiana. CSEP0027 contains a functional signal peptide (SP), verified by yeast secretion assay. We show that CSEP0027 promotes Bgh virulence in barley infection using transient gene expression and host-induced gene silencing (HIGS). Barley catalase HvCAT1 is identified as a CSEP0027 interactor by yeast two-hybrid (Y2H) screening, and the interaction is verified in yeast, in vitro and in vivo. The coexpression of CSEP0027 and HvCAT1 in barley cells results in altered localization of HvCAT1 from the peroxisome to the nucleus. Barley stripe mosaic virus (BSMV)-silencing and transiently-induced gene silencing (TIGS) assays reveal that HvCAT1 is required for barley immunity against Bgh. We propose that CSEP0027 interacts with barley HvCAT1 to regulate the host immunity and likely reactive oxygen species (ROS) homeostasis to promote fungal virulence during barley infection.

SnRK1 Phosphorylates and Destabilizes WRKY3 to Enhance Barley Immunity to Powdery Mildew.

Plants recognize pathogens and activate immune responses, which usually involve massive transcriptional reprogramming. The evolutionarily conserved kinase, Sucrose non-fermenting-related kinase 1 (SnRK1), functions as a metabolic regulator that is essential for plant growth and stress responses. Here, we identify barley SnRK1 and a WRKY3 transcription factor by screening a cDNA library. SnRK1 interacts with WRKY3 in yeast, as confirmed by pull-down and luciferase complementation assays. Förster resonance energy transfer combined with noninvasive fluorescence lifetime imaging analysis indicates that the interaction occurs in the barley nucleus. Transient expression and virus-induced gene silencing analyses indicate that WRKY3 acts as a repressor of disease resistance to the Bgh fungus. Barley plants overexpressing WRKY3 have enhanced fungal microcolony formation and sporulation. Phosphorylation assays show that SnRK1 phosphorylates WRKY3 mainly at Ser83 and Ser112 to destabilize the repressor, and WRKY3 non-phosphorylation-null mutants at these two sites are more stable than the wild-type protein. SnRK1-overexpressing barley plants display enhanced disease resistance to Bgh. Transient expression of SnRK1 reduces fungal haustorium formation in barley cells, which probably requires SnRK1 nuclear localization and kinase activity. Together, these findings suggest that SnRK1 is directly involved in plant immunity through phosphorylation and destabilization of the WRKY3 repressor, revealing a new regulatory mechanism of immune derepression in plants.

Proline isomerization of the immune receptor-interacting protein RIN4 by a cyclophilin inhibits effector-triggered immunity in Arabidopsis.

In the absence of pathogen infection, plant effector-triggered immune (ETI) receptors are maintained in a preactivation state by intermolecular interactions with other host proteins. Pathogen effector-induced alterations activate the receptor. In Arabidopsis, the ETI receptor RPM1 is activated via bacterial effector AvrB-induced phosphorylation of the RPM1-interacting protein RIN4 at Threonine 166. We find that RIN4 also interacts with the prolyl-peptidyl isomerase (PPIase) ROC1, which is reduced upon RIN4 Thr166 phosphorylation. ROC1 suppresses RPM1 immunity in a PPIase-dependent manner. Consistent with this, RIN4 Pro149 undergoes cis/trans isomerization in the presence of ROC1. While the RIN4(P149V) mutation abolishes RPM1 resistance, the deletion of Pro149 leads to RPM1 activation in the absence of RIN4 phosphorylation. These results support a model in which RPM1 directly senses conformational changes in RIN4 surrounding Pro149 that is controlled by ROC1. RIN4 Thr166 phosphorylation indirectly regulates RPM1 resistance by modulating the ROC1-mediated RIN4 isomerization.

Partitioning, repressing and derepressing: dynamic regulations in MLA immune receptor triggered defense signaling.

Plants and animals have evolved intracellular nucleotide-binding domain and leucine-rich repeat-containing immune receptors (NLRs) to perceive non-self and trigger immune responses. Plant NLRs detect strain-specific pathogen effectors and activate immune signaling leading to extensive transcriptional reprogramming and termination of pathogen infection. Here we review the recent findings in barley MLA immune receptor mediated immune responses against the barley powdery mildew fungus. We focus on nucleocytoplasmic partitioning of immune receptor, bifurcation of immune signaling, transcriptional repression and derepression connecting receptor activation to immune responses. We also discuss similar findings from other plant NLRs where appropriate.

Coiled-coil domain-dependent homodimerization of intracellular barley immune receptors defines a minimal functional module for triggering cell death.

Plants and animals have evolved structurally related innate immune sensors, designated NLRs, to detect intracellular nonself molecules. NLRs are modular, consisting of N-terminal coiled-coil (CC) or TOLL/interleukin-1 receptor (TIR) domains, a central nucleotide-binding (NB) domain, and C-terminal leucine-rich repeats (LRRs). The polymorphic barley mildew A (MLA) locus encodes CC-containing allelic immune receptors recognizing effectors of the pathogenic powdery mildew fungus. We report the crystal structure of an MLA receptor's invariant CC domain, which reveals a rod-shaped homodimer. MLA receptors also self-associate in vivo, but self-association appears to be independent of effector-triggered receptor activation. MLA CC mutants that fail to self-interact impair in planta cell death activity triggered by the CC domain alone and by an autoactive full-length MLA receptor that mimics its ATP-bound state. Thus, CC domain-dependent dimerization of the immune sensor defines a minimal functional unit and implies a role for the dimeric CC module in downstream immune signaling.

Nuclear activity of MLA immune receptors links isolate-specific and basal disease-resistance responses.

Plant immune responses are triggered by pattern recognition receptors that detect conserved pathogen-associated molecular patterns (PAMPs) or by resistance (R) proteins recognizing isolate-specific pathogen effectors. We show that in barley, intracellular mildew A (MLA) R proteins function in the nucleus to confer resistance against the powdery mildew fungus. Recognition of the fungal avirulence A10 effector by MLA10 induces nuclear associations between receptor and WRKY transcription factors. The identified WRKY proteins act as repressors of PAMP-triggered basal defense. MLA appears to interfere with the WRKY repressor function, thereby de-repressing PAMP-triggered basal defense. Our findings reveal a mechanism by which these polymorphic immune receptors integrate distinct pathogen signals.

Recognition specificity and RAR1/SGT1 dependence in barley Mla disease resistance genes to the powdery mildew fungus.

A large number of resistance specificities to the powdery mildew fungus Blumeria graminis f. sp. hordei map to the barley Mla locus. This complex locus harbors multiple members of three distantly related gene families that encode proteins that contain an N-terminal coiled-coil (CC) structure, a central nucleotide binding (NB) site, a Leu-rich repeat (LRR) region, and a C-terminal non-LRR (CT) region. We identified Mla12, which encodes a CC-NB-LRR-CT protein that shares 89 and 92% identical residues with the known proteins MLA1 and MLA6. Slow Mla12-triggered resistance was altered dramatically to a rapid response by overexpression of Mla12. A series of reciprocal domains swaps between MLA1 and MLA6 identified in each protein recognition domain for cognate powdery mildew fungus avirulence genes (AvrMla1 and AvrMla6). These domains were within different but overlapping LRR regions and the CT part. Unexpectedly, MLA chimeras that confer AvrMla6 recognition exhibited markedly different dependence on Rar1, a gene required for the function of some but not all Mla resistance specificities. Furthermore, uncoupling of MLA6-specific function from RAR1 also uncoupled the response from SGT1, a protein known to associate physically with RAR1. Our findings suggest that differences in the degree of RAR1 dependence of different MLA immunity responses are determined by intrinsic properties of MLA variants and place RAR1/SGT1 activity downstream of and/or coincident with the action of resistance protein-containing recognition complexes.

RAR1 positively controls steady state levels of barley MLA resistance proteins and enables sufficient MLA6 accumulation for effective resistance.

The polymorphic barley (Hordeum vulgare) Mla locus harbors allelic race-specific resistance (R) genes to the powdery mildew fungus Blumeria graminis f sp hordei. The highly sequence-related MLA proteins contain an N-terminal coiled-coil structure, a central nucleotide binding (NB) site, a Leu-rich repeat (LRR) region, and a C-terminal non-LRR region. Using transgenic barley lines expressing epitope-tagged MLA1 and MLA6 derivatives driven by native regulatory sequences, we show a reversible and salt concentration-dependent distribution of the intracellular MLA proteins in soluble and membrane-associated pools. A posttranscriptional process directs fourfold greater accumulation of MLA1 over MLA6. Unexpectedly, in rar1 mutant plants that are compromised for MLA6 but not MLA1 resistance, the steady state level of both MLA isoforms is reduced. Furthermore, differential steady state levels of MLA1/MLA6 hybrid proteins correlate with their requirement for RAR1; the RAR1-independent hybrid protein accumulates to higher levels and the RAR1-dependent one to lower levels. Interestingly, yeast two-hybrid studies reveal that the LRR domains of RAR1-independent but not RAR1-dependent MLA isoforms interact with SGT1, a RAR1 interacting protein required for the function of many NB-LRR type R proteins. Our findings implicate the existence of a conserved mechanism to reach minimal NB-LRR R protein thresholds that are needed to trigger effective resistance responses.