Phytophthora sojae effector Avr1d functions as an E2 competitor and inhibits ubiquitination activity of GmPUB13 to facilitate infection.

Oomycete pathogens such as Phytophthora secrete a repertoire of effectors into host cells to manipulate host immunity and benefit infection. In this study, we found that an RxLR effector, Avr1d, promoted Phytophthora sojae infection in soybean hairy roots. Using a yeast two-hybrid screen, we identified the soybean E3 ubiquitin ligase GmPUB13 as a host target for Avr1d. By coimmunoprecipitation (Co-IP), gel infiltration, and isothermal titration calorimetry (ITC) assays, we confirmed that Avr1d interacts with GmPUB13 both in vivo and in vitro. Furthermore, we found that Avr1d inhibits the E3 ligase activity of GmPUB13. The crystal structure Avr1d in complex with GmPUB13 was solved and revealed that Avr1d occupies the binding site for E2 ubiquitin conjugating enzyme on GmPUB13. In line with this, Avr1d competed with E2 ubiquitin conjugating enzymes for GmPUB13 binding in vitro, thereby decreasing the E3 ligase activity of GmPUB13. Meanwhile, we found that inactivation of the ubiquitin ligase activity of GmPUB13 stabilized GmPUB13 by blocking GmPUB13 degradation. Silencing of GmPUB13 in soybean hairy roots decreased P. sojae infection, suggesting that GmPUB13 acts as a susceptibility factor. Altogether, this study highlights a virulence mechanism of Phytophthora effectors, by which Avr1d competes with E2 for GmPUB13 binding to repress the GmPUB13 E3 ligase activity and thereby stabilizing the susceptibility factor GmPUB13 to facilitate Phytophthora infection. This study unravels the structural basis for modulation of host targets by Phytophthora effectors and will be instrumental for boosting plant resistance breeding.

Antagonistic modules regulate photosynthesis-associated nuclear genes via GOLDEN2-LIKE transcription factors.

GOLDEN2-LIKE (GLK) transcription factors drive the expression of photosynthesis-associated nuclear genes (PhANGs) indispensable for chloroplast biogenesis. Salicylic acid (SA)-induced SIGMA FACTOR-BINDING PROTEIN 1 (SIB1), a transcription coregulator and positive regulator of cell death, interacts with GLK1 and GLK2 to reinforce the expression of PhANGs, leading to photoinhibition of photosystem II and singlet oxygen (1O2) burst in chloroplasts. 1O2 then contributes to SA-induced cell death via EXECUTER 1 (EX1; 1O2 sensor protein)-mediated retrograde signaling upon reaching a critical level. This earlier finding has initiated research on the potential role of GLK1/2 and EX1 in SA signaling. Consistent with this view, we reveal that LESION-SIMULATING DISEASE 1 (LSD1), a transcription coregulator and negative regulator of SA-primed cell death, interacts with GLK1/2 to repress their activities in Arabidopsis (Arabidopsis thaliana). Overexpression of LSD1 repressed GLK target genes, including PhANGs, whereas loss of LSD1 enhanced their expression. Remarkably, LSD1 overexpression inhibited chloroplast biogenesis, resembling the characteristic glk1glk2 double mutant phenotype. Subsequent chromatin immunoprecipitation coupled with expression analyses further revealed that LSD1 inhibits the DNA-binding activity of GLK1 toward its target promoters. SA-induced nuclear-targeted SIB1 proteins appeared to interrupt the LSD1-GLK interaction, and the subsequent SIB1-GLK interaction activated EX1-mediated 1O2 signaling, elucidating antagonistic modules SIB1 and LSD1 in the regulation of GLK activity. Taken together, we provide a working model that SIB1 and LSD1, mutually exclusive SA-signaling components, antagonistically regulate GLK1/2 to fine-tune the expression of PhANGs, thereby modulating 1O2 homeostasis and related stress responses.

The Arabidopsis CRUMPLED LEAF protein, a homolog of the cyanobacterial bilin lyase, retains the bilin-binding pocket for a yet unknown function.

The photosynthetic bacterial phycobiliprotein lyases, also called CpcT lyases, catalyze the biogenesis of phycobilisome, a light-harvesting antenna complex, through the covalent attachment of chromophores to the antenna proteins. The Arabidopsis CRUMPLED LEAF (CRL) protein is a homolog of the cyanobacterial CpcT lyase. Loss of CRL leads to multiple lesions, including localized foliar cell death, constitutive expression of stress-related nuclear genes, abnormal cell cycle, and impaired plastid division. Notwithstanding the apparent phenotypes, the function of CRL still remains elusive. To gain insight into the function of CRL, we examined whether CRL still retains the capacity to bind with the bacterial chromophore phycocyanobilin (PCB) and its plant analog phytochromobilin (PΦB). The revealed structure of the CpcT domain of CRL is comparable to that of the CpcT lyase, despite the low sequence identity. The subsequent in vitro biochemical assays found, as shown for the CpcT lyase, that PCB/PΦB binds to the CRL dimer. However, some mutant forms of CRL, substantially compromised in their bilin-binding ability, still restore the crl-induced multiple lesions. These results suggest that although CRL retains the bilin-binding pocket, it seems not functionally associated with the crl-induced multiple lesions.

Glycolysis regulates pollen tube polarity via Rho GTPase signaling.

As a universal energy generation pathway utilizing carbon metabolism, glycolysis plays an important housekeeping role in all organisms. Pollen tubes expand rapidly via a mechanism of polarized growth, known as tip growth, to deliver sperm for fertilization. Here, we report a novel and surprising role of glycolysis in the regulation of growth polarity in Arabidopsis pollen tubes via impingement of Rho GTPase-dependent signaling. We identified a cytosolic phosphoglycerate kinase (pgkc-1) mutant with accelerated pollen germination and compromised pollen tube growth polarity. pgkc-1 mutation greatly diminished apical exocytic vesicular distribution of REN1 RopGAP (Rop GTPase activating protein), leading to ROP1 hyper-activation at the apical plasma membrane. Consequently, pgkc-1 pollen tubes contained higher amounts of exocytic vesicles and actin microfilaments in the apical region, and showed reduced sensitivity to Brefeldin A and Latrunculin B, respectively. While inhibition of mitochondrial respiration could not explain the pgkc-1 phenotype, the glycolytic activity is indeed required for PGKc function in pollen tubes. Moreover, the pgkc-1 pollen tube phenotype was mimicked by the inhibition of another glycolytic enzyme. These findings highlight an unconventional regulatory function for a housekeeping metabolic pathway in the spatial control of a fundamental cellular process.

A virus-targeted plant receptor-like kinase promotes cell-to-cell spread of RNAi.

RNA interference (RNAi) in plants can move from cell to cell, allowing for systemic spread of an antiviral immune response. How this cell-to-cell spread of silencing is regulated is currently unknown. Here, we describe that the C4 protein from Tomato yellow leaf curl virus can inhibit the intercellular spread of RNAi. Using this viral protein as a probe, we have identified the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1) as a positive regulator of the cell-to-cell movement of RNAi, and determined that BAM1 and its closest homolog, BAM2, play a redundant role in this process. C4 interacts with the intracellular domain of BAM1 and BAM2 at the plasma membrane and plasmodesmata, the cytoplasmic connections between plant cells, interfering with the function of these RLKs in the cell-to-cell spread of RNAi. Our results identify BAM1 as an element required for the cell-to-cell spread of RNAi and highlight that signaling components have been coopted to play multiple functions in plants.

Identification of endogenous small peptides involved in rice immunity through transcriptomics- and proteomics-based screening.

Small signalling peptides, generated from larger protein precursors, are important components to orchestrate various plant processes such as development and immune responses. However, small signalling peptides involved in plant immunity remain largely unknown. Here, we developed a pipeline using transcriptomics- and proteomics-based screening to identify putative precursors of small signalling peptides: small secreted proteins (SSPs) in rice, induced by rice blast fungus Magnaporthe oryzae and its elicitor, chitin. We identified 236 SSPs including members of two known small signalling peptide families, namely rapid alkalinization factors and phytosulfokines, as well as many other protein families that are known to be involved in immunity, such as proteinase inhibitors and pathogenesis-related protein families. We also isolated 52 unannotated SSPs and among them, we found one gene which we named immune response peptide (IRP) that appeared to encode the precursor of a small signalling peptide regulating rice immunity. In rice suspension cells, the expression of IRP was induced by bacterial peptidoglycan and fungal chitin. Overexpression of IRP enhanced the expression of a defence gene, PAL1 and induced the activation of the MAPKs in rice suspension cells. Moreover, the IRP protein level increased in suspension cell medium after chitin treatment. Collectively, we established a simple and efficient pipeline to discover SSP candidates that probably play important roles in rice immunity and identified 52 unannotated SSPs that may be useful for further elucidation of rice immunity. Our method can be applied to identify SSPs that are involved not only in immunity but also in other plant functions.

Impaired PSII Proteostasis Promotes Retrograde Signaling via Salicylic Acid.

Photodamage of the PSII reaction center (RC) is an inevitable process in an oxygen-rich environment. The damaged PSII RC proteins (Dam-PSII) undergo degradation via the thylakoid membrane-bound FtsH metalloprotease, followed by posttranslational assembly of PSII. While the effect of Dam-PSII on gene regulation is described for cyanobacteria, its role in land plants is largely unknown. In this study, we reveal an intriguing retrograde signaling pathway by using the Arabidopsis (Arabidopsis thaliana) yellow variegated2-9 mutant, which expresses a mutated FtsH2 (FtsH2G267D) metalloprotease, specifically impairing its substrate-unfolding activity. This lesion leads to the perturbation of PSII protein homeostasis (proteostasis) and the accumulation of Dam-PSII. Subsequently, this results in an up-regulation of salicylic acid (SA)-responsive genes, which is abrogated by inactivation of either an SA transporter in the chloroplast envelope membrane or extraplastidic SA signaling components as well as by removal of SA. These results suggest that the stress hormone SA, which is mainly synthesized via the chloroplast isochorismate pathway in response to the impaired PSII proteostasis, mediates the retrograde signaling. These findings reinforce the emerging view of chloroplast function toward plant stress responses and suggest SA as a potential plastid factor mediating retrograde signaling.

SCF(FBXL3) ubiquitin ligase targets cryptochromes at their cofactor pocket.

The cryptochrome (CRY) flavoproteins act as blue-light receptors in plants and insects, but perform light-independent functions at the core of the mammalian circadian clock. To drive clock oscillations, mammalian CRYs associate with the Period proteins (PERs) and together inhibit the transcription of their own genes. The SCF(FBXL3) ubiquitin ligase complex controls this negative feedback loop by promoting CRY ubiquitination and degradation. However, the molecular mechanisms of their interactions and the functional role of flavin adenine dinucleotide (FAD) binding in CRYs remain poorly understood. Here we report crystal structures of mammalian CRY2 in its apo, FAD-bound and FBXL3-SKP1-complexed forms. Distinct from other cryptochromes of known structures, mammalian CRY2 binds FAD dynamically with an open cofactor pocket. Notably, the F-box protein FBXL3 captures CRY2 by simultaneously occupying its FAD-binding pocket with a conserved carboxy-terminal tail and burying its PER-binding interface. This novel F-box-protein-substrate bipartite interaction is susceptible to disruption by both FAD and PERs, suggesting a new avenue for pharmacological targeting of the complex and a multifaceted regulatory mechanism of CRY ubiquitination.

Targeting Magnaporthe oryzae effector MoErs1 and host papain-like protease OsRD21 interaction to combat rice blast.

Effector proteins secreted by plant pathogenic fungi are important artilleries against host immunity, but there is no precedent of such effectors being explored as antifungal targets. Here we demonstrate that MoErs1, a species-specific effector protein secreted by the rice blast fungus Magnaporthe oryzae, inhibits the function of rice papain-like cysteine protease OsRD21 involved in rice immunity. Disrupting MoErs1-OsRD21 interaction effectively controls rice blast. In addition, we show that FY21001, a structure-function-based designer compound, specifically binds to and inhibits MoErs1 function. FY21001 significantly and effectively controls rice blast in field tests. Our study revealed a novel concept of targeting pathogen-specific effector proteins to prevent and manage crop diseases.

The structural basis for activation of plant immunity by bacterial effector protein AvrPto.

Pathogenic microbes use effectors to enhance susceptibility in host plants. However, plants have evolved a sophisticated immune system to detect these effectors using cognate disease resistance proteins, a recognition that is highly specific, often elicits rapid and localized cell death, known as a hypersensitive response, and thus potentially limits pathogen growth. Despite numerous genetic and biochemical studies on the interactions between pathogen effector proteins and plant resistance proteins, the structural bases for such interactions remain elusive. The direct interaction between the tomato protein kinase Pto and the Pseudomonas syringae effector protein AvrPto is known to trigger disease resistance and programmed cell death through the nucleotide-binding site/leucine-rich repeat (NBS-LRR) class of disease resistance protein Prf. Here we present the crystal structure of an AvrPto-Pto complex. Contrary to the widely held hypothesis that AvrPto activates Pto kinase activity, our structural and biochemical analyses demonstrated that AvrPto is an inhibitor of Pto kinase in vitro. The AvrPto-Pto interaction is mediated by the phosphorylation-stabilized P+1 loop and a second loop in Pto, both of which negatively regulate the Prf-mediated defences in the absence of AvrPto in tomato plants. Together, our results show that AvrPto derepresses host defences by interacting with the two defence-inhibition loops of Pto.

Pseudomonas syringae effector AvrPto blocks innate immunity by targeting receptor kinases.

Plants use receptor kinases, such as FLS2 and EFR, to perceive bacterial pathogens and initiate innate immunity. This immunity is often suppressed by bacterial effectors, allowing pathogen propagation. To counteract, plants have evolved disease resistance genes that detect the bacterial effectors and reinstate resistance. The Pseudomonas syringae effector AvrPto promotes infection in susceptible plants but triggers resistance in plants carrying the protein kinase Pto and the associated resistance protein Prf. Here we show that AvrPto binds receptor kinases, including Arabidopsis FLS2 and EFR and tomato LeFLS2, to block plant immune responses in the plant cell. The ability to target receptor kinases is required for the virulence function of AvrPto in plants. The FLS2-AvrPto interaction and Pto-AvrPto interaction appear to share similar sequence requirements, and Pto competes with FLS2 for AvrPto binding. The results suggest that the mechanism by which AvrPto recognizes virulence targets is linked to the evolution of Pto, which, in association with Prf, recognizes the bacterium and triggers strong resistance.

Expression, purification and crystallization of the N-terminal Solanaceae domain of the Sw-5b NLR immune receptor.

Plant nucleotide-binding domain and leucine-rich repeat receptors (NLRs) play crucial roles in recognizing pathogen effectors and activating plant immunity. The tomato NLR Sw-5b is a coiled-coil NLR (CC-NLR) immune receptor that confers resistance against tospoviruses, which cause serious economic losses in agronomic crops worldwide. Compared with other CC-NLRs, Sw-5b possesses an extended N-terminal Solanaceae domain (SD). The SD of Sw-5b is critical for recognition of the tospovirus viral movement protein NSm. An SD is also frequently detected in many NLRs from Solanaceae plants. However, no sequences homologous to the SD have been detected in animals or in plants other than Solanaceae. The properties of the SD protein are largely unknown, and thus 3D structural information is vital in order to better understand its role in pathogen perception and the activation of immune receptors. Here, the expression, purification and crystallization of Sw-5b SD (amino acids 1-245) are reported. Native and selenomethionine-substituted crystals of the SD protein belonged to space group P3112, with unit-cell parameters a = 81.53, b = 81.53, c = 98.44 Šand a = 81.63, b = 81.63, c = 98.80 Å, respectively. This is the first report of a structural study of the noncanonical SD domain of the NLR proteins from Solanaceae plants.

Mechanism of phosphate sensing and signaling revealed by rice SPX1-PHR2 complex structure.

Phosphate, a key plant nutrient, is perceived through inositol polyphosphates (InsPs) by SPX domain-containing proteins. SPX1 an inhibit the PHR2 transcription factor to maintain Pi homeostasis. How SPX1 recognizes an InsP molecule and represses transcription activation by PHR2 remains unclear. Here we show that, upon binding InsP6, SPX1 can disrupt PHR2 dimers and form a 1:1 SPX1-PHR2 complex. The complex structure reveals that SPX1 helix α1 can impose a steric hindrance when interacting with the PHR2 dimer. By stabilizing helix α1, InsP6 allosterically decouples the PHR2 dimer and stabilizes the SPX1-PHR2 interaction. In doing so, InsP6 further allows SPX1 to engage with the PHR2 MYB domain and sterically block its interaction with DNA. Taken together, our results suggest that, upon sensing the surrogate signals of phosphate, SPX1 inhibits PHR2 via a dual mechanism that attenuates dimerization and DNA binding activities of PHR2.

A phosphorylation-based switch controls TAA1-mediated auxin biosynthesis in plants.

Auxin determines the developmental fate of plant tissues, and local auxin concentration is precisely controlled. The role of auxin transport in modulating local auxin concentration has been widely studied but the regulation of local auxin biosynthesis is less well understood. Here, we show that TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA1), a key enzyme in the auxin biosynthesis pathway in Arabidopsis thaliana is phosphorylated at Threonine 101 (T101). T101 phosphorylation status can act as an on/off switch to control TAA1-dependent auxin biosynthesis and is required for proper regulation of root meristem size and root hair development. This phosphosite is evolutionarily conserved suggesting post-translational regulation of auxin biosynthesis may be a general phenomenon. In addition, we show that auxin itself, in part via TRANS-MEMBRANE KINASE 4 (TMK4), can induce T101 phosphorylation of TAA1 suggesting a self-regulatory loop whereby local auxin signalling can suppress biosynthesis. We conclude that phosphorylation-dependent control of TAA1 enzymatic activity may contribute to regulation of auxin concentration in response to endogenous and/or external cues.

PAWH1 and PAWH2 are plant-specific components of an Arabidopsis endoplasmic reticulum-associated degradation complex.

Endoplasmic reticulum-associated degradation (ERAD) is a unique mechanism to degrade misfolded proteins via complexes containing several highly-conserved ER-anchored ubiquitin ligases such as HMG-CoA reductase degradation1 (Hrd1). Arabidopsis has a similar Hrd1-containing ERAD machinery; however, our knowledge of this complex is limited. Here we report two closely-related Arabidopsis proteins, Protein Associated With Hrd1-1 (PAWH1) and PAWH2, which share a conserved domain with yeast Altered Inheritance of Mitochondria24. PAWH1 and PAWH2 localize to the ER membrane and associate with Hrd1 via EMS-mutagenized Bri1 Suppressor7 (EBS7), a plant-specific component of the Hrd1 complex. Simultaneously elimination of two PAWHs constitutively activates the unfolded protein response and compromises stress tolerance. Importantly, the pawh1 pawh2 double mutation reduces the protein abundance of EBS7 and Hrd1 and inhibits degradation of several ERAD substrates. Our study not only discovers additional plant-specific components of the Arabidopsis Hrd1 complex but also reveals a distinct mechanism for regulating the Hrd1 stability.

Phytophthora sojae Effector PsAvh240 Inhibits Host Aspartic Protease Secretion to Promote Infection.

Plants secrete defense molecules into the extracellular space (the apoplast) to combat attacking microbes. However, the mechanisms by which successful pathogens subvert plant apoplastic immunity remain poorly understood. In this study, we show that PsAvh240, a membrane-localized effector of the soybean pathogen Phytophthora sojae, promotes P. sojae infection in soybean hairy roots. We found that PsAvh240 interacts with the soybean-resistant aspartic protease GmAP1 in planta and suppresses the secretion of GmAP1 into the apoplast. By solving its crystal structure we revealed that PsAvh240 contain six α helices and two WY motifs. The first two α helices of PsAvh240 are responsible for its plasma membrane-localization and are required for PsAvh240's interaction with GmAP1. The second WY motifs of two PsAvh240 molecules form a handshake arrangement resulting in a handshake-like dimer. This dimerization is required for the effector's repression of GmAP1 secretion. Taken together, these data reveal that PsAvh240 localizes at the plasma membrane to interfere with GmAP1 secretion, which represents an effective mechanism by which effector proteins suppress plant apoplastic immunity.

An oomycete plant pathogen reprograms host pre-mRNA splicing to subvert immunity.

The process of RNA splicing influences many physiological processes, including plant immunity. However, how plant parasites manipulate host RNA splicing process remains unknown. Here we demonstrate that PsAvr3c, an avirulence effector from oomycete plant pathogen Phytophthora sojae, physically binds to and stabilizes soybean serine/lysine/arginine-rich proteins GmSKRPs. The SKRPs are novel proteins that associate with a complex that contains plant spliceosome components, and are negative regulators of plant immunity. Analysis by RNA-seq data indicates that alternative splicing of pre-mRNAs from 401 soybean genes, including defense-related genes, is altered in GmSKRP1 and PsAvr3c overexpressing lines compared to control plants. Representative splicing events mediated by GmSKRP1 and PsAvr3c are tested by infection assays or by transient expression in soybean plants. Our results show that plant pathogen effectors can reprogram host pre-mRNA splicing to promote disease, and we propose that pathogens evolved such strategies to defeat host immune systems.

2.6 A resolution crystal structure of the bacterioferritin from Azotobacter vinelandii.

The crystal structure of the bacterioferritin from Azotobacter vinelandii has been determined at 2.6 A resolution. Both the low occupancy of one iron ion in the dinuclear iron center and the deviation of its adjacent residue His130 from the center suggest migration of the iron ion from the dinuclear iron site to the inner nucleation site. The concerted movement of His130 and Glu47 may admit a dynamic gating mechanism for shift of the oxidized iron ion. Ba2+ binding to the fourfold channel implicates that the channel bears Fe2+ conductivity and selectivity to provide a route for iron access to the inner cavity during core formation.