Root Endophyte Colletotrichum tofieldiae Confers Plant Fitness Benefits that Are Phosphate Status Dependent.

A staggering diversity of endophytic fungi associate with healthy plants in nature, but it is usually unclear whether these represent stochastic encounters or provide host fitness benefits. Although most characterized species of the fungal genus Colletotrichum are destructive pathogens, we show here that C. tofieldiae (Ct) is an endemic endophyte in natural Arabidopsis thaliana populations in central Spain. Colonization by Ct initiates in roots but can also spread systemically into shoots. Ct transfers the macronutrient phosphorus to shoots, promotes plant growth, and increases fertility only under phosphorus-deficient conditions, a nutrient status that might have facilitated the transition from pathogenic to beneficial lifestyles. The host's phosphate starvation response (PSR) system controls Ct root colonization and is needed for plant growth promotion (PGP). PGP also requires PEN2-dependent indole glucosinolate metabolism, a component of innate immune responses, indicating a functional link between innate immunity and the PSR system during beneficial interactions with Ct.

The Arabidopsis SGN3/GSO1 receptor kinase integrates soil nitrogen status into shoot development.

The Casparian strip is a barrier in the endodermal cell walls of plants that allows the selective uptake of nutrients and water. In the model plant Arabidopsis thaliana, its development and establishment are under the control of a receptor-ligand mechanism termed the Schengen pathway. This pathway facilitates barrier formation and activates downstream compensatory responses in case of dysfunction. However, due to a very tight functional association with the Casparian strip, other potential signaling functions of the Schengen pathway remain obscure. In this work, we created a MYB36-dependent synthetic positive feedback loop that drives Casparian strip formation independently of Schengen-induced signaling. We evaluated this by subjecting plants in which the Schengen pathway has been uncoupled from barrier formation, as well as a number of established barrier-mutant plants, to agar-based and soil conditions that mimic agricultural settings. Under the latter conditions, the Schengen pathway is necessary for the establishment of nitrogen-deficiency responses in shoots. These data highlight Schengen signaling as an essential hub for the adaptive integration of signaling from the rhizosphere to aboveground tissues.

Three-Dimensional Ultrastructure of Arabidopsis Cotyledons Infected with Colletotrichum higginsianum.

We used serial block-face scanning electron microscopy (SBF-SEM) to study the host-pathogen interface between Arabidopsis cotyledons and the hemibiotrophic fungus Colletotrichum higginsianum. By combining high-pressure freezing and freeze-substitution with SBF-SEM, followed by segmentation and reconstruction of the imaging volume using the freely accessible software IMOD, we created 3D models of the series of cytological events that occur during the Colletotrichum-Arabidopsis susceptible interaction. We found that the host cell membranes underwent massive expansion to accommodate the rapidly growing intracellular hypha. As the fungal infection proceeded from the biotrophic to the necrotrophic stage, the host cell membranes went through increasing levels of disintegration culminating in host cell death. Intriguingly, we documented autophagosomes in proximity to biotrophic hyphae using transmission electron microscopy (TEM) and a concurrent increase in autophagic flux between early to mid/late biotrophic phase of the infection process. Occasionally, we observed osmiophilic bodies in the vicinity of biotrophic hyphae using TEM only and near necrotrophic hyphae under both TEM and SBF-SEM. Overall, we established a method for obtaining serial SBF-SEM images, each with a lateral (x-y) pixel resolution of 10 nm and an axial (z) resolution of 40 nm, that can be reconstructed into interactive 3D models using the IMOD. Application of this method to the Colletotrichum-Arabidopsis pathosystem allowed us to more fully understand the spatial arrangement and morphological architecture of the fungal hyphae after they penetrate epidermal cells of Arabidopsis cotyledons and the cytological changes the host cell undergoes as the infection progresses toward necrotrophy. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

The development of pleiotropic phenotypes in powdery mildew-resistant barley and Arabidopsis thaliana mlo mutants is linked to nitrogen availability.

Powdery mildew-resistant barley (Hordeum vulgare) and Arabidopsis thaliana mlo mutant plants exhibit pleiotropic phenotypes such as the spontaneous formation of callose-rich cell wall appositions and early leaf chlorosis and necrosis, indicative of premature leaf senescence. The exogenous factors governing the occurrence of these undesired side effects remain poorly understood. Here, we characterised the formation of these symptoms in detail. Ultrastructural analysis revealed that the callose-rich cell wall depositions spontaneously formed in A. thaliana mlo mutants are indistinguishable from those induced by the bacterial pattern epitope, flagellin 22 (flg22). We further found that increased plant densities during culturing enhance the extent of the leaf senescence syndrome in A. thaliana mlo mutants. Application of a liquid fertiliser rescued the occurrence of leaf chlorosis and necrosis in both A. thaliana and barley mlo mutant plants. Controlled fertilisation experiments uncovered nitrogen as the macronutrient whose deficiency promotes the extent of pleiotropic phenotypes in A. thaliana mlo mutants. Light intensity and temperature had a modulatory impact on the incidence of leaf necrosis in the case of barley mlo mutant plants. Collectively, our data indicate that the development of pleiotropic phenotypes associated with mlo mutants is governed by various exogenous factors.

A high-confidence Physcomitrium patens plasmodesmata proteome by iterative scoring and validation reveals diversification of cell wall proteins during evolution.

Plasmodesmata (PD) facilitate movement of molecules between plant cells. Regulation of this movement is still not understood. Plasmodesmata are hard to study, being deeply embedded within cell walls and incorporating several membrane types. Thus, structure and protein composition of PD remain enigmatic. Previous studies of PD protein composition identified protein lists with few validations, making functional conclusions difficult. We developed a PD scoring approach in iteration with large-scale systematic localization, defining a high-confidence PD proteome of Physcomitrium patens (HC300). HC300, together with bona fide PD proteins from literature, were placed in Pddb. About 65% of proteins in HC300 were not previously PD-localized. Callose-degrading glycolyl hydrolase family 17 (GHL17) is an abundant protein family with representatives across evolutionary scale. Among GHL17s, we exclusively found members of one phylogenetic clade with PD localization and orthologs occur only in species with developed PD. Phylogenetic comparison was expanded to xyloglucan endotransglucosylases/hydrolases and Exordium-like proteins, which also diversified into PD-localized and non-PD-localized members on distinct phylogenetic clades. Our high-confidence PD proteome HC300 provides insights into diversification of large protein families. Iterative and systematic large-scale localization across plant species strengthens the reliability of HC300 as basis for exploring structure, function, and evolution of this important organelle.

FLOWERING REPRESSOR AAA+ ATPase 1 is a novel regulator of perennial flowering in Arabis alpina.

Arabis alpina is a polycarpic perennial, in which PERPETUAL FLOWERING1 (PEP1) regulates flowering and perennial traits in a vernalization-dependent manner. Mutagenesis screens of the pep1 mutant established the role of other flowering time regulators in PEP1-parallel pathways. Here we characterized three allelic enhancers of pep1 (eop002, 085 and 091) which flower early. We mapped the causal mutations and complemented mutants with the identified gene. Using quantitative reverse transcriptase PCR and reporter lines, we determined the protein spatiotemporal expression patterns and localization within the cell. We also characterized its role in Arabidopsis thaliana using CRISPR and in A. alpina by introgressing mutant alleles into a wild-type background. These mutants carried lesions in an AAA+ ATPase of unknown function, FLOWERING REPRESSOR AAA+ ATPase 1 (AaFRAT1). AaFRAT1 was detected in the vasculature of young leaf primordia and the rib zone of flowering shoot apical meristems. At the subcellular level, AaFRAT1 was localized at the interphase between the endoplasmic reticulum and peroxisomes. Introgression lines carrying Aafrat1 alleles required less vernalization to flower and reduced number of vegetative axillary branches. By contrast, A. thaliana CRISPR lines showed weak flowering phenotypes. AaFRAT1 contributes to flowering time regulation and the perennial growth habit of A. alpina.

Beyond flowering time: diverse roles of an APETALA2-like transcription factor in shoot architecture and perennial traits.

Polycarpic perennials maintain vegetative growth after flowering. PERPETUAL FLOWERING 1 (PEP1), the orthologue of FLOWERING LOCUS C (FLC) in Arabis alpina regulates flowering and contributes to polycarpy in a vernalisation-dependent pathway. pep1 mutants do not require vernalisation to flower and have reduced return to vegetative growth as all of their axillary branches become reproductive. To identify additional genes that regulate flowering and contribute to perennial traits we performed an enhancer screen of pep1. Using mapping-by-sequencing, we cloned a mutant (enhancer of pep1-055, eop055), performed transcriptome analysis and physiologically characterised the role it plays on perennial traits in an introgression line carrying the eop055 mutation and a functional PEP1 wild-type allele. eop055 flowers earlier than pep1 and carries a lesion in the A. alpina orthologue of the APETALA2 (AP2)-like gene, TARGET OF EAT2 (AaTOE2). AaTOE2 is a floral repressor and acts upstream of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 5 (AaSPL5). In the wild-type background, which requires cold treatment to flower, AaTOE2 regulates the age-dependent response to vernalisation. In addition, AaTOE2 ensures the maintenance of vegetative growth by delaying axillary meristem initiation and repressing flowering of axillary buds before and during cold exposure. We conclude that AaTOE2 is instrumental in fine tuning different developmental traits in the perennial life cycle of A. alpina.

Chloroplast Acetyltransferase NSI Is Required for State Transitions in Arabidopsis thaliana.

The amount of light energy received by the photosynthetic reaction centers photosystem II (PSII) and photosystem I (PSI) is balanced through state transitions. Reversible phosphorylation of a light-harvesting antenna trimer (L-LHCII) orchestrates the association between L-LHCII and the photosystems, thus adjusting the amount of excitation energy received by the reaction centers. In this study, we identified the enzyme NUCLEAR SHUTTLE INTERACTING (NSI; AT1G32070) as an active lysine acetyltransferase in the chloroplasts of Arabidopsis thaliana Intriguingly, nsi knockout mutant plants were defective in state transitions, even though they had a similar LHCII phosphorylation pattern as the wild type. Accordingly, nsi plants were not able to accumulate the PSI-LHCII state transition complex, even though the LHCII docking site of PSI and the overall amounts of photosynthetic protein complexes remained unchanged. Instead, the nsi mutants showed a decreased Lys acetylation status of specific photosynthetic proteins including PSI, PSII, and LHCII subunits. Our work demonstrates that the chloroplast acetyltransferase NSI is needed for the dynamic reorganization of thylakoid protein complexes during photosynthetic state transitions.

Vernalization shapes shoot architecture and ensures the maintenance of dormant buds in the perennial Arabis alpina.

Perennials have a complex shoot architecture with axillary meristems organized in zones of differential bud activity and fate. This includes zones of buds maintained dormant for multiple seasons and used as reservoirs for potential growth in case of damage. The shoot of Arabis alpina, a perennial relative of Arabidopsis thaliana, consists of a zone of dormant buds placed between subapical vegetative and basal flowering branches. This shoot architecture is shaped after exposure to prolonged cold, required for flowering. To understand how vernalization ensures the maintenance of dormant buds, we performed physiological and transcriptome studies, followed the spatiotemporal changes of auxin, and generated transgenic plants. Our results demonstrate that the complex shoot architecture in A. alpina is shaped by its flowering behavior, specifically the initiation of inflorescences during cold treatment and rapid flowering after subsequent exposure to growth-promoting conditions. Dormant buds are already formed before cold treatment. However, dormancy in these buds is enhanced during, and stably maintained after, vernalization by a BRC1-dependent mechanism. Post-vernalization, stable maintenance of dormant buds is correlated with increased auxin response, transport, and endogenous indole-3-acetic acid levels in the stem. Here, we provide a functional link between flowering and the maintenance of dormant buds in perennials.

Seed coat development in explosively dispersed seeds of Cardamine hirsuta.

BACKGROUND AND AIMS: Seeds are dispersed by explosive coiling of the fruit valves in Cardamine hirsuta. This rapid coiling launches the small seeds on ballistic trajectories to spread over a 2 m radius around the parent plant. The seed surface interacts with both the coiling fruit valve during launch and subsequently with the air during flight. We aim to identify features of the seed surface that may contribute to these interactions by characterizing seed coat differentiation. METHODS: Differentiation of the outermost seed coat layers from the outer integuments of the ovule involves dramatic cellular changes that we characterize in detail at the light and electron microscopical level including immunofluorescence and immunogold labelling. KEY RESULTS: We found that the two outer integument (oi) layers of the seed coat contributed differently to the topography of the seed surface in the explosively dispersed seeds of C. hirsuta vs. the related species Arabidopsis thaliana where seed dispersal is non-explosive. The surface of A. thaliana seeds is shaped by the columella and the anticlinal cell walls of the epidermal oi2 layer. In contrast, the surface of C. hirsuta seeds is shaped by a network of prominent ridges formed by the anticlinal walls of asymmetrically thickened cells of the sub-epidermal oi1 layer, especially at the seed margin. Both the oi2 and oi1 cell layers in C. hirsuta seeds are characterized by specialized, pectin-rich cell walls that are deposited asymmetrically in the cell. CONCLUSIONS: The two outermost seed coat layers in C. hirsuta have distinct properties: the sub-epidermal oi1 layer determines the topography of the seed surface, while the epidermal oi2 layer accumulates mucilage. These properties are influenced by polar deposition of distinct pectin polysaccharides in the cell wall. Although the ridged seed surface formed by oi1 cell walls is associated with ballistic dispersal in C. hirsuta, it is not restricted to explosively dispersed seeds in the Brassicaceae.

FGB1 and WSC3 are in planta-induced ß-glucan-binding fungal lectins with different functions.

In the root endophyte Serendipita indica, several lectin-like members of the expanded multigene family of WSC proteins are transcriptionally induced in planta and are potentially involved in ß-glucan remodeling at the fungal cell wall. Using biochemical and cytological approaches we show that one of these lectins, SiWSC3 with three WSC domains, is an integral fungal cell wall component that binds to long-chain ß1-3-glucan but has no affinity for shorter ß1-3- or ß1-6-linked glucose oligomers. Comparative analysis with the previously identified ß-glucan-binding lectin SiFGB1 demonstrated that whereas SiWSC3 does not require ß1-6-linked glucose for efficient binding to branched ß1-3-glucan, SiFGB1 does. In contrast to SiFGB1, the multivalent SiWSC3 lectin can efficiently agglutinate fungal cells and is additionally induced during fungus-fungus confrontation, suggesting different functions for these two ß-glucan-binding lectins. Our results highlight the importance of the ß-glucan cell wall component in plant-fungus interactions and the potential of ß-glucan-binding lectins as specific detection tools for fungi in vivo.

Colletotrichum higginsianum extracellular LysM proteins play dual roles in appressorial function and suppression of chitin-triggered plant immunity.

The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large repertoire of candidate-secreted effectors containing LysM domains, but the role of such proteins in the pathogenicity of any Colletotrichum species is unknown. Here, we characterized the function of two effectors, ChELP1 and ChELP2, which are transcriptionally activated during the initial intracellular biotrophic phase of infection. Using immunocytochemistry, we found that ChELP2 is concentrated on the surface of bulbous biotrophic hyphae at the interface with living host cells but is absent from filamentous necrotrophic hyphae. We show that recombinant ChELP1 and ChELP2 bind chitin and chitin oligomers in vitro with high affinity and specificity and that both proteins suppress the chitin-triggered activation of two immune-related plant mitogen-activated protein kinases in the host Arabidopsis. Using RNAi-mediated gene silencing, we found that ChELP1 and ChELP2 are essential for fungal virulence and appressorium-mediated penetration of both Arabidopsis epidermal cells and cellophane membranes in vitro. The findings suggest a dual role for these LysM proteins as effectors for suppressing chitin-triggered immunity and as proteins required for appressorium function.

The powdery mildew resistance protein RPW8.2 is carried on VAMP721/722 vesicles to the extrahaustorial membrane of haustorial complexes.

Plants employ multiple cell-autonomous defense mechanisms to impede pathogenesis of microbial intruders. Previously we identified an exocytosis defense mechanism in Arabidopsis against pathogenic powdery mildew fungi. This pre-invasive defense mechanism depends on the formation of ternary protein complexes consisting of the plasma membrane-localized PEN1 syntaxin, the adaptor protein SNAP33 and closely sequence-related vesicle-resident VAMP721 or VAMP722 proteins. The Arabidopsis thaliana resistance to powdery mildew 8.2 protein (RPW8.2) confers disease resistance against powdery mildews upon fungal entry into host cells and is specifically targeted to the extrahaustorial membrane (EHM), which envelops the haustorial complex of the fungus. However, the secretory machinery involved in trafficking RPW8.2 to the EHM is unknown. Here we report that RPW8.2 is transiently located on VAMP721/722 vesicles, and later incorporated into the EHM of mature haustoria. Resistance activity of RPW8.2 against the powdery mildew Golovinomyces orontii is greatly diminished in the absence of VAMP721 but only slightly so in the absence of VAMP722. Consistent with this result, trafficking of RPW8.2 to the EHM is delayed in the absence of VAMP721. These findings implicate VAMP721/722 vesicles as key components of the secretory machinery for carrying RPW8.2 to the plant-fungal interface. Quantitative fluorescence recovery after photobleaching suggests that vesicle-mediated trafficking of RPW8.2-yellow fluorescent protein (YFP) to the EHM occurs transiently during early haustorial development and that lateral diffusion of RPW8.2-YFP within the EHM exceeds vesicle-mediated replenishment of RPW8.2-YFP in mature haustoria. Our findings imply the engagement of VAMP721/722 in a bifurcated trafficking pathway for pre-invasive defense at the cell periphery and post-invasive defense at the EHM.

Sieve element occlusion (SEO) genes encode structural phloem proteins involved in wound sealing of the phloem.

The sieve element occlusion (SEO) gene family originally was delimited to genes encoding structural components of forisomes, which are specialized crystalloid phloem proteins found solely in the Fabaceae. More recently, SEO genes discovered in various non-Fabaceae plants were proposed to encode the common phloem proteins (P-proteins) that plug sieve plates after wounding. We carried out a comprehensive characterization of two tobacco (Nicotiana tabacum) SEO genes (NtSEO). Reporter genes controlled by the NtSEO promoters were expressed specifically in immature sieve elements, and GFP-SEO fusion proteins formed parietal agglomerates in intact sieve elements as well as sieve plate plugs after wounding. NtSEO proteins with and without fluorescent protein tags formed agglomerates similar in structure to native P-protein bodies when transiently coexpressed in Nicotiana benthamiana, and the analysis of these protein complexes by electron microscopy revealed ultrastructural features resembling those of native P-proteins. NtSEO-RNA interference lines were essentially devoid of P-protein structures and lost photoassimilates more rapidly after injury than control plants, thus confirming the role of P-proteins in sieve tube sealing. We therefore provide direct evidence that SEO genes in tobacco encode P-protein subunits that affect translocation. We also found that peptides recently identified in fascicular phloem P-protein plugs from squash (Cucurbita maxima) represent cucurbit members of the SEO family. Our results therefore suggest a common evolutionary origin for P-proteins found in the sieve elements of all dicotyledonous plants and demonstrate the exceptional status of extrafascicular P-proteins in cucurbits.

TIME FOR COFFEE is an essential component in the maintenance of metabolic homeostasis in Arabidopsis thaliana.

Plants often respond to environmental changes by reprogramming metabolic and stress-associated pathways. Homeostatic integration of signaling is a central requirement for ensuring metabolic stability in living organisms. Under diurnal conditions, properly timed rhythmic metabolism provides fitness benefits to plants. TIME FOR COFFEE (TIC) is a circadian regulator known to be involved in clock resetting at dawn. Here we explored the mechanism of influence of TIC in plant growth and development, as initiated by a microarray analysis. This global profiling showed that a loss of TIC function causes a major reprogramming of gene expression that predicts numerous developmental, metabolic, and stress-related phenotypes. This led us to demonstrate that this mutant exhibits late flowering, a plastochron defect, and diverse anatomical phenotypes. We further observed a starch-excess phenotype and altered soluble carbohydrate levels. tic exhibited hypersensitivity to oxidative stress and abscisic acid, and this was associated with a striking resistance to drought. These phenotypes were connected to an increase in total glutathione levels that correlated with a readjustment of amino acids and polyamine pools. By comparatively analyzing our transcriptomic and metabolomic data, we concluded that TIC is a central element in plant homeostasis that integrates and coordinates developmental, metabolic, and environmental signals.

Sequential delivery of host-induced virulence effectors by appressoria and intracellular hyphae of the phytopathogen Colletotrichum higginsianum.

Phytopathogens secrete effector proteins to manipulate their hosts for effective colonization. Hemibiotrophic fungi must maintain host viability during initial biotrophic growth and elicit host death for subsequent necrotrophic growth. To identify effectors mediating these opposing processes, we deeply sequenced the transcriptome of Colletotrichum higginsianum infecting Arabidopsis. Most effector genes are host-induced and expressed in consecutive waves associated with pathogenic transitions, indicating distinct effector suites are deployed at each stage. Using fluorescent protein tagging and transmission electron microscopy-immunogold labelling, we found effectors localised to stage-specific compartments at the host-pathogen interface. In particular, we show effectors are focally secreted from appressorial penetration pores before host invasion, revealing new levels of functional complexity for this fungal organ. Furthermore, we demonstrate that antagonistic effectors either induce or suppress plant cell death. Based on these results we conclude that hemibiotrophy in Colletotrichum is orchestrated through the coordinated expression of antagonistic effectors supporting either cell viability or cell death.