Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism.

In flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling, an inducer of the synergid nuclear disorganization. Therefore, two female gametes (the egg and the central cell) control independent pathways yet coordinately accomplish the elimination of the persistent synergid cell by double fertilization.

Chromatin phosphoproteomics unravels a function for AT-hook motif nuclear localized protein AHL13 in PAMP-triggered immunity.

In many eukaryotic systems during immune responses, mitogen-activated protein kinases (MAPKs) link cytoplasmic signaling to chromatin events by targeting transcription factors, chromatin remodeling complexes, and the RNA polymerase machinery. So far, knowledge on these events is scarce in plants and no attempts have been made to focus on phosphorylation events of chromatin-associated proteins. Here we carried out chromatin phosphoproteomics upon elicitor-induced activation of Arabidopsis The events in WT were compared with those in mpk3, mpk4, and mpk6 mutant plants to decipher specific MAPK targets. Our study highlights distinct signaling networks involving MPK3, MPK4, and MPK6 in chromatin organization and modification, as well as in RNA transcription and processing. Among the chromatin targets, we characterized the AT-hook motif containing nuclear localized (AHL) DNA-binding protein AHL13 as a substrate of immune MAPKs. AHL13 knockout mutant plants are compromised in pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species production, expression of defense genes, and PAMP-triggered immunity. Transcriptome analysis revealed that AHL13 regulates key factors of jasmonic acid biosynthesis and signaling and affects immunity toward Pseudomonas syringae and Botrytis cinerea pathogens. Mutational analysis of the phosphorylation sites of AHL13 demonstrated that phosphorylation regulates AHL13 protein stability and thereby its immune functions.

Essential role of the CD docking motif of MPK4 in plant immunity, growth, and development.

MAPKs are universal eukaryotic signaling factors whose functioning is assumed to depend on the recognition of a common docking motif (CD) by its activators, substrates, and inactivators. We studied the role of the CD domain of Arabidopsis MPK4 by performing interaction studies and determining the ligand-bound MPK4 crystal structure. We revealed that the CD domain of MPK4 is essential for interaction and activation by its upstream MAPKKs MKK1, MKK2, and MKK6. Cys181 in the CD site of MPK4 was shown to become sulfenylated in response to reactive oxygen species in vitro. To test the function of C181 in vivo, we generated wild-type (WT) MPK4-C181, nonsulfenylatable MPK4-C181S, and potentially sulfenylation mimicking MPK4-C181D lines in the mpk4 knockout background. We analyzed the phenotypes in growth, development, and stress responses, revealing that MPK4-C181S has WT activity and complements the mpk4 phenotype. By contrast, MPK4-C181D cannot be activated by upstream MAPKK and cannot complement the phenotypes of mpk4. Our findings show that the CD motif is essential and is required for activation by upstream MAPKK for MPK4 function. Furthermore, growth, development, or immunity functions require upstream activation of the MPK4 protein kinase.

The rice/maize pathogen Cochliobolus spp. infect and reproduce on Arabidopsis revealing differences in defensive phytohormone function between monocots and dicots.

The fungal genus Cochliobolus describes necrotrophic pathogens that give rise to significant losses on rice, wheat, and maize. Revealing plant mechanisms of non-host resistance (NHR) against Cochliobolus will help to uncover strategies that can be exploited in engineered cereals. Therefore, we developed a heterogeneous pathosystem and studied the ability of Cochliobolus to infect dicotyledons. We report here that C. miyabeanus and C. heterostrophus infect Arabidopsis accessions and produce functional conidia, thereby demonstrating the ability to accept Brassica spp. as host plants. Some ecotypes exhibited a high susceptibility, whereas others hindered the necrotrophic disease progression of the Cochliobolus strains. Natural variation in NHR among the tested Arabidopsis accessions can advance the identification of genetic loci that prime the plant's defence repertoire. We found that applied phytotoxin-containing conidial fluid extracts of C. miyabeanus caused necrotic lesions on rice leaves but provoked only minor irritations on Arabidopsis. This result implies that C. miyabeanus phytotoxins are insufficiently adapted to promote dicot colonization, which corresponds to a retarded infection progression. Previous studies on rice demonstrated that ethylene (ET) promotes C. miyabeanus infection, whereas salicylic acid (SA) and jasmonic acid (JA) exert a minor function. However, in Arabidopsis, we revealed that the genetic disruption of the ET and JA signalling pathways compromises basal resistance against Cochliobolus, whereas SA biosynthesis mutants showed a reduced susceptibility. Our results refer to the synergistic action of ET/JA and indicate distinct defence systems between Arabidopsis and rice to confine Cochliobolus propagation. Moreover, this heterogeneous pathosystem may help to reveal mechanisms of NHR and associated defensive genes against Cochliobolus infection.

Lyso-phosphatidylethanolamine primes the plant immune system and promotes basal resistance against hemibiotrophic pathogens.

BACKGROUND: Lyso-phosphatidylethanolamine (LPE) is a natural phospholipid that functions in the early stages of plant senescence. Plant innate immunity and early leaf senescence share molecular components. To reveal conserved mechanisms that link-up both processes, we tried to unravel to what extent LPE coordinates defense response and by what mode of action. RESULT: We found that LPE-treatment induces signaling and biosynthesis gene expression of the defensive hormone salicylic acid (SA). However, jasmonic acid and ethylene triggered gene induction levels are indistinguishable from the control. In accordance with gene induction for SA, oxidative stress, and reactive oxygen species (ROS) production, we detected raised in-situ hydrogen peroxide levels following LPE-application. Yet, ROS-burst assays of LPE-pretreated plants revealed a reduced release of ROS after PAMP-administration suggesting that LPE interferes with an oxidative burst. Our data refer to a priming effect of LPE on SA/ROS-associated genomic loci that encode pivotal factors in early senescence and considerably improve plant basal immunity. Thus, we challenged Arabidopsis thaliana with the hemibiotrophic pathogen Pseudomonas syringae. Consistently, we found an increased resistance in the LPE-pretreated Arabidopsis plants compared to the mock-pretreated control. CONCLUSIONS: Our results underscore a beneficial effect of LPE on plant innate immunity against hemibiotrophs. Given the resistance-promoting effect of exogenously applied LPE, this bio-agent bears the potential of being applied as a valuable tool for the genetic activation of defense-associated traits.

INDETERMINATE-DOMAIN 4 (IDD4) coordinates immune responses with plant-growth in Arabidopsis thaliana.

INDETERMINATE DOMAIN (IDD)/ BIRD proteins are a highly conserved plant-specific family of transcription factors which play multiple roles in plant development and physiology. Here, we show that mutation in IDD4/IMPERIAL EAGLE increases resistance to the hemi-biotrophic pathogen Pseudomonas syringae, indicating that IDD4 may act as a repressor of basal immune response and PAMP-triggered immunity. Furthermore, the idd4 mutant exhibits enhanced plant-growth indicating IDD4 as suppressor of growth and development. Transcriptome comparison of idd4 mutants and IDD4ox lines aligned to genome-wide IDD4 DNA-binding studies revealed major target genes related to defense and developmental-biological processes. IDD4 is a phospho-protein that interacts and becomes phosphorylated on two conserved sites by the MAP kinase MPK6. DNA-binding studies of IDD4 after flg22 treatment and with IDD4 phosphosite mutants show enhanced binding affinity to ID1 motif-containing promoters and its function as a transcriptional regulator. In contrast to the IDD4-phospho-dead mutant, the IDD4 phospho-mimicking mutant shows altered susceptibility to PstDC3000, salicylic acid levels and transcriptome reprogramming. In summary, we found that IDD4 regulates various hormonal pathways thereby coordinating growth and development with basal immunity.

The Trihelix transcription factor GT2-like 1 (GTL1) promotes salicylic acid metabolism, and regulates bacterial-triggered immunity.

The Trihelix Transcription factor GT2-like 1 (GTL1) was previously shown to be a key regulator of ploidy-dependent trichome growth and drought tolerance. Here, we report that GTL1 plays an important role in coordinating plant immunity. We show that gtl1 mutants are compromised in the regulation of basal immunity, microbial pattern-triggered immunity (PTI) and effector-triggered RIN4-mediated immunity. Transcriptome analysis revealed that GTL1 positively regulates defense genes and inhibits factors that mediate growth and development. By performing hormonal measurements and chromatin-immunoprecipitation studies, we found GTL1 to coordinate genes involved in salicylic acid metabolism, transport and response. Interaction studies and comparative transcriptomics to known data sets revealed that GTL1 is part of the MPK4 pathway and regulates oppositely the expression of differentially expressed genes in mpk4 plants. We introduced the gtl1 mutation in the mpk4 mutant and thereby partially suppressed its dwarfism and the high resistance against a bacterial invader. Our data show that GTL1 is part of the MPK4 pathway and acts as a positive regulator of bacterial-triggered immunity and SA homeostasis.

LACHESIS-dependent egg-cell signaling regulates the development of female gametophytic cells.

In contrast to animals, plant germ cells are formed along with accessory cells in specialized haploid generations, termed gametophytes. The female gametophyte of flowering plants consists of four different cell types, which exert distinct functions in the reproductive process. For successful fertilization, the development of the four cell types has to be tightly coordinated; however, the underlying mechanisms are not yet understood. We have previously isolated the lachesis (lis) mutant, which forms supernumerary gametes at the expense of adjacent accessory cells. LIS codes for the Arabidopsis homolog of the pre-mRNA splicing factor PRP4 and shows a dynamic expression pattern in the maturing female gametophyte. Here, we used LIS as a molecular tool to study cell-cell communication in the female gametophyte. We show that reducing LIS transcript amounts specifically in the egg cell, affects the development of all female gametophytic cells, indicating that cell differentiation in the female gametophyte is orchestrated by the egg cell. Among the defects observed is the failure of homotypic nuclei fusion in the central cell and, as a consequence, a block in endosperm formation. LIS-mediated egg cell signaling, thus, provides a safeguard mechanism that prevents the formation of nurturing tissue in the absence of a functional egg cell.

Chloroplast-related host proteins interact with NIb and NIa-Pro of soybeans mosaic virus and induce resistance in the susceptible cultivar.

To gain a deeper understanding of the molecular mechanisms involved in viral infection and the corresponding plant resistance responses, it is essential to investigate the interactions between viral and host proteins. In the case of viral infections in plants, a significant portion of the affected gene products are closely associated with chloroplasts and photosynthesis. However, the molecular mechanisms underlying the interplay between the virus and host chloroplast proteins during replication remain poorly understood. In our previous study, we made an interesting discovery regarding soybean mosaic virus (SMV) infection in resistant and susceptible soybean cultivars. We found that the photosystem I (PSI) subunit (PSaC) and ATP synthase subunit α (ATPsyn-α) genes were up-regulated in the resistant cultivar following SMV-G7H and SMV-G5H infections compared to the susceptible cultivar. Overexpression of these two genes within the SMV-G7H genome in the susceptible cultivar Lee74 (rsv3-null) reduced SMV accumulation, whereas silencing of the PSaC and ATPsyn-α genes promoted SMV accumulation. We have also found that the PSaC and ATPsyn-α proteins are present in the chloroplast envelope, nucleus, and cytoplasm. Building on these findings, we now characterized protein-protein interactions between PSaC and ATPsyn-α with two viral proteins, NIb and NIa-Pro, respectively, of SMV. Through co-immunoprecipitation (Co-IP) experiments, we confirmed the interactions between these proteins. Moreover, when the C-terminal region of either PSaC or ATPsyn-α was overexpressed in the SMV-G7H genome, we observed a reduction in viral accumulation and systemic infection in the susceptible cultivar. Based on these results, we propose that the PSaC and ATPsyn-α genes play a modulatory role in conferring resistance to SMV infection by influencing the function of NIb and NIa-Pro-in SMV replication and movement. The identification of these photosynthesis-related genes as key players in the interplay between the virus and the host provides valuable insights for developing more targeted control strategies against SMV. Additionally, by utilizing these genes, it may be possible to genetically engineer plants with improved photosynthetic efficiency and enhanced resistance to SMV infection.

The nuclear effector MoHTR3 of Magnaporthe oryzae modulates host defence signalling in the biotrophic stage of rice infection.

Fungal effectors play a pivotal role in suppressing the host defence system, and their evolution is highly dynamic. By comparative sequence analysis of plant-pathogenic fungi and Magnaporthe oryzae, we identified the small secreted C2 H2 zinc finger protein MoHTR3. MoHTR3 exhibited high conservation in M. oryzae strains but low conservation among other plant-pathogenic fungi, suggesting an emerging evolutionary selection process. MoHTR3 is exclusively expressed in the biotrophic stage of fungal invasion, and the encoded protein localizes to the biotrophic interfacial complex (BIC) and the host cell nucleus. The signal peptide crucial for MoHTR3' secretion to the BIC and the protein section required for its translocation to the nucleus were both identified by a functional protein domain study. The host-nuclear localization of MoHTR3 suggests a function as a transcriptional modulator of host defence gene induction. After ΔMohtr3 infection, the expression of jasmonic acid- and ethylene-associated genes was diminished in rice, in contrast to when the MoHTR3-overexpressing strain (MoHTR3ox) was applied. The transcript levels of salicylic acid- and defence-related genes were also affected after ΔMohtr3 and MoHTR3ox application. In pathogenicity assays, ΔMohtr3 was indistinguishable from the wild type. However, MoHTR3ox-infected plants showed diminished lesion formation and hydrogen peroxide accumulation, accompanied by a decrease in susceptibility, suggesting that the MoHTR3-induced manipulation of host cells affects host-pathogen interaction. MoHTR3 emphasizes the role of the host nucleus as a critical target for the pathogen-driven manipulation of host defence mechanisms and underscores the ongoing evolution of rice blast's arms race.

Salinity stress-induced phosphorylation of INDETERMINATE-DOMAIN 4 (IDD4) by MPK6 regulates plant growth adaptation in Arabidopsis.

The INDETERMINATE DOMAIN (IDD) family belongs to a group of plant-specific transcription factors that coordinates plant growth/development and immunity. However, the function and mode of action of IDDs during abiotic stress, such as salt, are poorly understood. We used idd4 transgenic lines and screened them under salt stress to find the involvement of IDD4 in salinity stress tolerance The genetic disruption of IDD4 increases salt-tolerance, characterized by sustained plant growth, improved Na+/K+ ratio, and decreased stomatal density/aperture. Yet, IDD4 overexpressing plants were hypersensitive to salt-stress with an increase in stomatal density and pore size. Transcriptomic and ChIP-seq analyses revealed that IDD4 directly controls an important set of genes involved in abiotic stress/salinity responses. Interestingly, using anti-IDD4-pS73 antibody we discovered that IDD4 is specifically phosphorylated at serine-73 by MPK6 in vivo under salinity stress. Analysis of plants expressing the phospho-dead and phospho-mimicking IDD4 versions proved that phosphorylation of IDD4 plays a crucial role in plant transcriptional reprogramming of salt-stress genes. Altogether, we show that salt stress adaption involves MPK6 phosphorylation of IDD4 thereby regulating IDD4 DNA-binding and expression of target genes.

ROS homeostasis mediated by MPK4 and SUMM2 determines synergid cell death.

Sexual plant reproduction depends on the attraction of sperm-cell delivering pollen tubes (PT) by two synergids, followed by their programmed cell death (PCD) in Arabidopsis. Disruption of the mitogen-activated protein kinase 4 (MPK4) by pathogenic effectors activates the resistance protein (R) SUMM2-mediated immunity and cell death. Here we show that synergid preservation and reactive oxygen species (ROS) homeostasis are intimately linked and maintained by MPK4. In mpk4, ROS levels are increased and synergids prematurely undergo PCD before PT-reception. However, ROS scavengers and the disruption of SUMM2, in mpk4, restore ROS homeostasis, synergid maintenance and PT perception, demonstrating that the guardian of MPK4, SUMM2, triggers synergid-PCD. In mpk4/summ2, PTs show a feronia-like overgrowth phenotype. Our results show that immunity-associated PCD and synergid cell death during plant reproduction are regulated by MPK4 underscoring an underlying molecular mechanism for the suppression of plant reproduction during systemic R-mediated immunity.

Dual and opposing roles of EIN3 reveal a generation conflict during seed growth.

Seed size critically affects grain yield of crops and hence represents a key breeding target. The development of embryo-nourishing endosperm is a key driver of seed expansion. We here report unexpected dual roles of the transcription factor EIN3 in regulating seed size. These EIN3 functions have remained largely undiscovered because they oppose each other. Capitalizing on the analysis of multiple ethylene biosynthesis mutants, we demonstrate that EIN3 represses endosperm and seed development in a pathway regulated by ethylene. We, in addition, provide evidence that EIN3-mediated synergid nucleus disintegration promotes endosperm expansion. Interestingly, synergid nucleus disintegration is not affected in various ethylene biosynthesis mutants, suggesting that this promoting function of EIN3 is independent of ethylene. Whereas the growth-inhibitory ethylene-dependent EIN3 action appears to be encoded by sporophytic tissue, the growth-promoting role of EIN3 is induced by fertilization, revealing a generation conflict that converges toward the key signaling component EIN3.

Characterization of the MYB Genes Reveals Insights Into Their Evolutionary Conservation, Structural Diversity, and Functional Roles in Magnaporthe oryzae.

The myeloblastosis (MYB) transcription factor family is evolutionarily conserved among plants, animals, and fungi, and contributes to their growth and development. We identified and analyzed 10 putative MYB genes in Magnaporthe oryzae (MoMYB) and determined their phylogenetic relationships, revealing high divergence and variability. Although MYB domains are generally defined by three tandem repeats, MoMYBs contain one or two weakly conserved repeats embedded in extensive disordered regions. We characterized the secondary domain organization, disordered segments, and functional contributions of each MoMYB. During infection, MoMYBs are distinctively expressed and can be subdivided into two clades of being either up- or down-regulated. Among these, MoMYB1 and MoMYB8 are up-regulated during infection and vegetative growth, respectively. We found MoMYB1 localized predominantly to the cytosol during the formation of infection structures. ΔMomyb1 exhibited reduced virulence on intact rice leaves corresponding to the diminished ability to form hypha-driven appressorium (HDA). We discovered that MoMYB1 regulates HDA formation on hard, hydrophobic surfaces, whereas host surfaces partially restored HDA formation in ΔMomyb1. Lipid droplet accumulation in hyphal tips and expression of HDA-associated genes were strongly perturbed in ΔMomyb1 indicating genetic interaction of MoMYB1 with downstream components critical to HDA formation. We also found that MoMYB8 is necessary for fungal growth, dark-induced melanization of hyphae, and involved in higher abiotic stress tolerance. Taken together, we revealed a multifaceted picture of the MoMYB family, wherein a low degree of conservation has led to the development of distinct structures and functions, ranging from fungal growth to virulence.

Phosphorylation regulates the activity of INDETERMINATE-DOMAIN (IDD/BIRD) proteins in response to diverse environmental conditions.

INDETERMINATE-DOMAIN proteins (IDDs) belong to a diverse plant-specific family of transcriptional regulators that coordinate distinct functions during plant growth and development. The functions of several of these IDD members are transcriptionally regulated, but so far nothing is known about the regulation at the post-translational level in spite of the fact that post-translational modifications of these proteins have been reported in several large-scale proteomics studies. Recently, we showed that IDD4 is a repressor of basal immunity and its characteristic traits are predominantly determined by the phosphorylation at two distinct phosphorylation sites. This finding prompted us to comprehensively review phosphorylation of the various IDD members from the plethora of phosphoproteomics studies demonstrating the post-translational modification of IDDs at highly conserved sites under various experimental conditions. We reckon that the phosphorylation of IDDs is an underrated mechanistic aspect in their regulation and we postulate their importance in IDD/BIRD functioning.

Ethylene signaling is required for synergid degeneration and the establishment of a pollen tube block.

In flowering plants, sperm cells are delivered by pollen tubes, which are attracted by two egg-cell-adjoining synergids. Successful fertilization terminates pollen tube attraction; however, the underlying mechanisms are not understood. Here, we show that the process of fertilization activates an EIN3- and EIN2-dependent ethylene-response cascade necessary for synergid cell death and the concomitant establishment of a pollen tube block. Microinjection of the ethylene precursor ACC into the female gametophyte or constitutive ethylene response results in premature synergid disintegration. This indicates that the requirement of fertilization for synergid degeneration and associated establishment of a pollen tube block can be bypassed by mimicking a postfertilization ethylene burst. Surprisingly, the persistent synergid in ethylene-hyposensitive plants adopts the molecular profile and cell-cycle regime of the biparental embryo-nourishing tissue, suggesting that ethylene signaling prevents the formation of an asexual maternal endosperm fraction.

Female gametophytic mutants: diagnosis and characterization.

In plants, gametes are formed in multicellular haploid structures, termed gametophytes. The female gametophyte of most higher plants comprises seven cells, which develop from a single haploid spore through nuclear proliferation and subsequent cellularization. The female gametophytic cells differentiate into four distinct cell types, which play specific roles during fertilization and seed formation thereby ensuring reproductive success. In recent years many new techniques and cell type-specific marker lines have been established, making the female gametophyte an attractive system to study mechanisms of reproduction as well as cell specification. The following chapter describes a basic protocol for, first of all, recognizing a female gametophytic mutant and subsequently analyzing the phenotype on a morphological, molecular, and functional level.