Lignin accumulation in cell wall plays a role in clubroot resistance.

Clubroot, caused by Plasmodiophora brassicae, is a significant disease affecting brassica crops worldwide and poses a threat to canola (Brassica napus) production in western Canada. Management of this disease heavily relies on the use of resistant cultivars, but resistance erosion is a serious concern due to the highly diverse pathogen populations. Understanding resistance mechanisms may aid in better deployment/rotation of clubroot resistance (CR) genes and improve resistance resilience. In this study, we conducted a comparative analysis using resistant canola varieties carrying either a single (Rcr1) or double CR genes (Rcr1+Crr1rutb ) to decipher the resistance modes associated with these genes. Cell wall (CW) biopolymeric compounds in different root layers were mapped and quantified using Fourier-transform mid-infrared microspectroscopy for changes in CW elements associated with clubroot resistance. Transmission electron and confocal microscopy were used to assess root infection details and relative transcript abundance was analyzed to determine the activation of the lignin-related pathway in relation to resistance. Neither resistant variety affected the primary infection of root hairs/epidermal cells compared to the susceptible "Westar", but both exhibited strong inhibition of cortical infection, effectively 'trapping' the pathogen in the exodermis. The most prominent change observed was increased lignin accumulation associated with resistance. In Westar, the pathogen was able to degrade CW lignin, facilitating access to the root cortex by secondary plasmodia of P. brassicae. In contrast, resistant varieties showed clear lignin accumulation around the penetration site on the exodermis, accompanied by elevated expression of genes involved in the phenylpropanoid pathway. These results suggest that induced lignin accumulation plays a role in clubroot resistance mediated by the CR genes Rcr1 and Crr1rutb in canola, providing cellular and structural evidence that supports the data from earlier transcriptomic studies.

The Plasmodiophora brassicae Golgi-localized UPF0016 protein PbGDT1 mediates calcium but not manganese transport in yeast and Nicotiana benthamiana.

Manganese and calcium homeostasis and signalling, in eukaryotic organisms, are regulated through membrane located pumps, channels and exchangers, including the Mn2+/Ca2+ uncharacterized protein family 0016 (UPF0016). Here we show that Plasmodiophora brassicae PbGDT1 is a member of the UPF0016 and an ortholog of Saccharomyces cerevisiae Gdt1p (GCR Dependent Translation Factor 1) protein involved in manganese homeostasis as well as the calcium mediated stress response in yeast. PbGDT1 complemented the ScGdt1p and ScPMR1 (Ca2+ ATPase) double null mutant under elevated calcium stress but not under elevated manganese conditions. In both yeast and Nicotiana benthamiana, PbGDT1 localizes to the Golgi apparatus, with additional ER association in N. benthamiana. Expression of PbGDT1 in N. benthamiana, suppresses BAX-triggered cell death, further highlighting the importance of calcium homeostasis in maintaining cell physiology and integrity in a stress environment.

The Gß-like protein Bcgbl1 regulates development and pathogenicity of the gray mold Botrytis cinerea via modulating two MAP kinase signaling pathways.

The fungal Gß-like protein has been reported to be involved in a variety of biological processes, such as mycelial growth, differentiation, conidiation, stress responses and infection. However, molecular mechanisms of the Gß-like protein in regulating fungal development and pathogenicity are largely unknown. Here, we show that the Gß-like protein gene Bcgbl1 in the gray mold fungus Botrytis cinerea plays a pivotal role in development and pathogenicity by regulating the mitogen-activated protein (MAP) kinases signaling pathways. The Bcgbl1 deletion mutants were defective in mycelial growth, sclerotial formation, conidiation, macroconidial morphogenesis, plant adhesion, and formation of infection cushions and appressorium-like structures, resulting in a complete loss of pathogenicity. Bcgbl1 interacted with BcSte50, the adapter protein of the cascade of MAP kinase (MAPK). Bcgbl1 mutants had reduced phosphorylation levels of two MAPKs, namely Bmp1 and Bmp3, thereby reducing infection. However, deletion of Bcgbl1 did not affect the intracellular cAMP level, and exogenous cAMP could not restore the defects. Moreover, Bcgbl1 mutants exhibited defects in cell wall integrity and oxidative stress tolerance. Transcriptional profiling revealed that Bcgbl1 plays a global role in regulation of gene expression upon hydrophobic surface induction. We further uncovered that three target genes encoding the hydrophobic surface binding proteins (HsbAs) contributed to the adhesion and virulence of B. cinerea. Overall, these findings suggest that Bcgbl1 had multiple functions and provided new insights for deciphering the Bcgbl1-mediated network for regulating development and pathogenicity of B. cinerea.

Expression of a mycoparasite protease in plant petals suppresses the petal-mediated infection by necrotrophic pathogens.

Sclerotinia sclerotiorum and Botrytis cinerea are necrotrophic plant-pathogenic fungi, causing substantial economic losses on many crops. So far, resistant cultivars against these pathogens are unavailable in most crops. Here, we show that the serine protease CmSp1 of Coniothyrium minitans, a well-characterized mycoparasite of S. sclerotiorum, contributed to suppressing the petal-mediated infection by S. sclerotiorum in rapeseed. Application of recombinant CmSp1 proteins facilitates the bulk degradation of S. sclerotiorum proteins and inhibits spore germination and hyphal growth of S. sclerotiorum and B. cinerea, thereby preventing the development of both diseases. Stable transgenic rapeseed plants with tissue-specific expression of CmSp1 in flower petals inhibit the petal-mediated infection by both S. sclerotiorum and B. cinerea, and resulting transgenic plants have no adverse effect on other agronomic traits. Thus, our findings provide a novel mechanism by which a mycoparasite inhibits fungal pathogens and an environmentally friendly disease management strategy.

Spatiotemporal transcriptomics and metabolic profiling provide insights into gene regulatory networks during lentil seed development.

Lentil (Lens culinaris Medik.) is a nutritious legume with seeds rich in protein, minerals and an array of diverse specialized metabolites. The formation of a seed requires regulation and tight coordination of developmental programs to form the embryo, endosperm and seed coat compartments, which determines the structure and composition of mature seed and thus its end-use quality. Understanding the molecular and cellular events and metabolic processes of seed development is essential for improving lentil yield and seed nutritional value. However, such information remains largely unknown, especially at the seed compartment level. In this study, we generated high-resolution spatiotemporal gene expression profiles in lentil embryo, seed coat and whole seeds from fertilization through maturation. Apart from anatomic differences between the embryo and seed coat, comparative transcriptomics and weighted gene co-expression network analysis revealed embryo- and seed coat-specific genes and gene modules predominant in specific tissues and stages, which highlights distinct genetic programming. Furthermore, we investigated the dynamic profiles of flavonoid, isoflavone, phytic acid and saponin in seed compartments across seed development. Coupled with transcriptome data, we identified sets of candidate genes involved in the biosynthesis of these metabolites. The global view of the transcriptional and metabolic changes of lentil seed tissues throughout development provides a valuable resource for dissecting the genetic control of secondary metabolism and development of molecular tools for improving seed nutritional quality.

The plant trans-Golgi network component ECHIDNA regulates defense, cell death, and endoplasmic reticulum stress.

The trans-Golgi network (TGN) acts as a central platform for sorting and secreting various cargoes to the cell surface, thus being essential for the full execution of plant immunity. However, the fine-tuned regulation of TGN components in plant defense and stress response has been not fully elucidated. Our study revealed that despite largely compromising penetration resistance, the loss-of-function mutation of the TGN component protein ECHIDNA (ECH) induced enhanced postinvasion resistance to powdery mildew in Arabidopsis thaliana. Genetic and transcriptome analyses and hormone profiling demonstrated that ECH loss resulted in salicylic acid (SA) hyperaccumulation via the ISOCHORISMATE SYNTHASE 1 biosynthesis pathway, thereby constitutively activating SA-dependent innate immunity that was largely responsible for the enhanced postinvasion resistance. Furthermore, the ech mutant displayed accelerated SA-independent spontaneous cell death and constitutive POWDERY MILDEW RESISTANCE 4-mediated callose depositions. In addition, ECH loss led to a chronically prolonged endoplasmic reticulum stress in the ech mutant. These results provide insights into understanding the role of TGN components in the regulation of plant immunity and stress responses.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9-generated diallelic mutants reveal Arabidopsis actin-related protein 2 function in the trafficking of syntaxin PEN1.

In plants, the actin cytoskeleton plays a critical role in defense against diverse pathogens. The formation of actin patches is essential for the intracellular transport of organelles and molecules toward pathogen penetration sites and the formation of papillae for an early cellular response to powdery mildew attack in Arabidopsis thaliana. This response process is regulated by the actin-related protein (ARP)2/3 complex and its activator, the WAVE/SCAR complex (W/SRC). The ARP2/3 complex is also required for maintaining steady-state levels of the defense-associated protein, PENETRATION 1 (PEN1), at the plasma membrane and for its deposition into papillae. However, specific ARP2 functionalities in this context remain unresolved, as knockout mutants expressing GFP-PEN1 reporter constructs could not be obtained by conventional crossing approaches. In this study, employing a CRISPR/Cas9 multiplexing-mediated genome editing approach, we produced an ARP2 knockout expressing the GFP-PEN1 marker in Arabidopsis. This study successfully identified diallelic somatic mutations with both ARP2 alleles edited among the primary T1 transgenic plants, and also obtained independent lines with stable arp2/arp2 mutations in the T2 generation. Further analyses on these arp2/arp2 mutants showed similar biological functions of ARP2 to ARP3 in the accumulation of PEN1 against fungal invasion. Together, this CRISPR/Cas9-based approach offers highly efficient simultaneous disruption of the two ARP2 alleles in GFP-PEN1-expressing lines, and a rapid method for performing live-cell imaging to facilitate the investigation of important plant-pathogen interactions using a well-established and widely applied GFP marker system, thus gaining insights and elucidating the contributions of ARP2 upon fungal attack.

Cold and exogenous calcium alter Allium fistulosum cell wall pectin to depress intracellular freezing temperatures.

De-methyl esterification of homogalacturonan and subsequent cross-linking with Ca2+ is hypothesized to enhance the freezing survival of cold acclimated plants by reducing the porosity of primary cell walls. To test this theory, we collected leaf epidermal peels from non- (23/18 °C) and cold acclimated (2 weeks at 12/4 °C) Japanese bunching onion (Allium fistulosum L.). Cold acclimation enhanced the temperature at which half the cells survived freezing injury by 8 °C (LT50 =-20 °C), and reduced tissue permeability by 70-fold compared with non-acclimated epidermal cells. These effects were associated with greater activity of pectin methylesterase (PME) and a reduction in the methyl esterification of homogalacturonan. Non-acclimated plants treated with 50 mM CaCl2 accumulated higher concentrations of galacturonic acid, Ca2+ in the cell wall, and a lower number of visible cell wall pores compared with that observed in cold acclimated plants. Using cryo-microscopy, we observed that 50 mM CaCl2 treatment did not lower the LT50 of non-acclimated cells, but reduced the lethal intracellular ice nucleation to temperatures observed in cold acclimated epidermal cells. We postulate that the PME-homogalacturonan-mediated reduction in cell wall porosity is integral to intracellular freezing avoidance strategies in cold acclimated herbaceous cells.

With a Little Help from My Cell Wall: Structural Modifications in Pectin May Play a Role to Overcome Both Dehydration Stress and Fungal Pathogens.

Cell wall structural modifications through pectin cross-linkages between calcium ions and/or boric acid may be key to mitigating dehydration stress and fungal pathogens. Water loss was profiled in a pure pectin system and in vivo. While calcium and boron reduced water loss in pure pectin standards, the impact on Allium species was insignificant (p > 0.05). Nevertheless, synchrotron X-ray microscopy showed the localization of exogenously applied calcium to the apoplast in the epidermal cells of Allium fistulosum. Exogenous calcium application increased viscosity and resistance to shear force in Allium fistulosum, suggesting the formation of calcium cross-linkages ("egg-box" structures). Moreover, Allium fistulosum (freezing tolerant) was also more tolerant to dehydration stress compared to Allium cepa (freezing sensitive). Furthermore, the addition of boric acid (H3BO3) to pure pectin reduced water loss and increased viscosity, which indicates the formation of RG-II dimers. The Arabidopsis boron transport mutant, bor1, expressed greater water loss and, based on the lesion area of leaf tissue, a greater susceptibility to Colletotrichum higginsianum and Botrytis cinerea. While pectin modifications in the cell wall are likely not the sole solution to dehydration and biotic stress resistance, they appear to play an important role against multiple stresses.

Evolutionary divergence in embryo and seed coat development of U's Triangle Brassica species illustrated by a spatiotemporal transcriptome atlas.

The economically valuable Brassica species include the six related members of U's Triangle. Despite the agronomic and economic importance of these Brassicas, the impacts of evolution and relatively recent domestication events on the genetic landscape of seed development have not been comprehensively examined in these species. Here we present a 3D transcriptome atlas for the six species of U's Triangle, producing a unique resource that captures gene expression data for the major subcompartments of the seed, from the unfertilized ovule to the mature embryo and seed coat. This comprehensive dataset for seed development in tetraploid and ancestral diploid Brassicas provides new insights into evolutionary divergence and expression bias at the gene and subgenome levels during the domestication of these valued crop species. Comparisons of gene expression associated with regulatory networks and metabolic pathways operating in the embryo and seed coat during seed development reveal differences in storage reserve accumulation and fatty acid metabolism among the six Brassica species. This study illustrates the genetic underpinnings of seed traits and the selective pressures placed on seed production, providing an immense resource for continued investigation of Brassica polyploid biology, genomics and evolution.

A clubroot pathogen effector targets cruciferous cysteine proteases to suppress plant immunity.

Plant pathogen effector proteins are key to pathogen virulence. In susceptible host Brassicas, the clubroot pathogen, Plasmodiophora brassicae, induces the production of nutrient-sink root galls, at the site of infection. Among a list of 32 P. brassiae effector candidates previously reported by our group, we identified SSPbP53 as a putative apoplastic cystatin-like protein highly expressed during the secondary infection. Here we found that SSPbP53 encoding gene is conserved among several P. brassicae pathotypes and that SSPbP53 is an apoplastic protein able to directly interact with and inhibit cruciferous papain-like cysteine proteases (PLCPs), specifically Arabidopsis XYLEM CYSTEINE PEPTIDASE 1 (AtXCP1). The severity of clubroot disease is greatly reduced in the Arabidopsis xcp1 null mutant (AtΔxcp1) after infection with P. brassicae resting spores, indicating that the interaction of P. brassicae SSPbP53 with XCP1 is important to clubroot susceptibility. SSPbP53 is the first cystatin-like effector identified and characterized for a plant pathogenic protist.

Endomembrane-Targeting Plasmodiophora brassicae Effectors Modulate PAMP Triggered Immune Responses in Plants.

Plasmodiophora brassicae is a devastating obligate, intracellular, biotrophic pathogen that causes clubroot disease in crucifer plants. Disease progression is regulated by effector proteins secreted by P. brassicae. Twelve P. brassicae putative effectors (PbPEs), expressed at various stages of disease development [0, 2, 5, 7, 14, 21, and 28 days post inoculation (DPI)] in Arabidopsis and localizing to the plant endomembrane system, were studied for their roles in pathogenesis. Of the 12 PbPEs, seven showed an inhibitory effect on programmed cell death (PCD) as triggered by the PCD inducers, PiINF1 (Phytophthora infestans Infestin 1) and PiNPP1 (P. infestans necrosis causing protein). Showing the strongest level of PCD suppression, PbPE15, a member of the 2-oxoglutarate (2OG) and Fe (II)-dependent oxygenase superfamily and with gene expression during later stages of infection, appears to have a role in tumorigenesis as well as defense signaling in plants. PbPE13 produced an enhanced PiINF1-induced PCD response. Transient expression, in Nicotiana benthamiana leaves of these PbPEs minus the signal peptide (SP) (Δsp PbPEGFPs), showed localization to the endomembrane system, targeting the endoplasmic reticulum (ER), Golgi bodies and nucleo-cytoplasm, suggesting roles in manipulating plant cell secretion and vesicle trafficking. Δsp PbPE13GFP localized to plasma membrane (PM) lipid rafts with an association to plasmodesmata, suggesting a role at the cell-to-cell communication junction. Membrane relocalization of Δsp PbPE13GFP, triggered by flagellin N-terminus of Pseudomonas aeruginosa (flg22 - known to elicit a PAMP triggered immune response in plants), supports its involvement in raft-mediated immune signaling. This study is an important step in deciphering P. brassicae effector roles in the disruption of plant immunity to clubroot disease.

The ARP2/3 complex, acting cooperatively with Class I formins, modulates penetration resistance in Arabidopsis against powdery mildew invasion.

The actin cytoskeleton regulates an array of diverse cellular activities that support the establishment of plant-microbe interactions and plays a critical role in the execution of plant immunity. However, molecular and cellular mechanisms regulating the assembly and rearrangement of actin filaments (AFs) at plant-pathogen interaction sites remain largely elusive. Here, using live-cell imaging, we show that one of the earliest cellular responses in Arabidopsis thaliana upon powdery mildew attack is the formation of patch-like AF structures beneath fungal invasion sites. The AFs constituting actin patches undergo rapid turnover, which is regulated by the actin-related protein (ARP)2/3 complex and its activator, the WAVE/SCAR regulatory complex (W/SRC). The focal accumulation of phosphatidylinositol-4,5-bisphosphate at fungal penetration sites appears to be a crucial upstream modulator of the W/SRC-ARP2/3 pathway-mediated actin patch formation. Knockout of W/SRC-ARP2/3 pathway subunits partially compromised penetration resistance with impaired endocytic recycling of the defense-associated t-SNARE protein PEN1 and its deposition into apoplastic papillae. Simultaneously knocking out ARP3 and knocking down the Class I formin (AtFH1) abolished actin patch formation, severely impaired the deposition of cell wall appositions, and promoted powdery mildew entry into host cells. Our results demonstrate that the ARP2/3 complex and formins, two actin-nucleating systems, act cooperatively and contribute to Arabidopsis penetration resistance to fungal invasion.

Alternative splicing dynamics and evolutionary divergence during embryogenesis in wheat species.

Among polyploid species with complex genomic architecture, variations in the regulation of alternative splicing (AS) provide opportunities for transcriptional and proteomic plasticity and the potential for generating trait diversities. However, the evolution of AS and its influence on grain development in diploid grass and valuable polyploid wheat crops are poorly understood. To address this knowledge gap, we developed a pipeline for the analysis of alternatively spliced transcript isoforms, which takes the high sequence similarity among polyploid wheat subgenomes into account. Through analysis of synteny and detection of collinearity of homoeologous subgenomes, conserved and specific AS events across five wheat and grass species were identified. A global analysis of the regulation of AS in diploid grass and polyploid wheat grains revealed diversity in AS events not only between the endosperm, pericarp and embryo overdevelopment, but also between subgenomes. Analysis of AS in homoeologous triads of polyploid wheats revealed evolutionary divergence between gene-level and transcript-level regulation of embryogenesis. Evolutionary age analysis indicated that the generation of novel transcript isoforms has occurred in young genes at a more rapid rate than in ancient genes. These findings, together with the development of comprehensive AS resources for wheat and grass species, advance understanding of the evolution of regulatory features of AS during embryogenesis and grain development in wheat.

Comparing the Infection Biology of Plasmodiophora brassicae in Clubroot Susceptible and Resistant Hosts and Non-hosts.

The potential infection biology of Plasmodiophora brassicae in resistant hosts and non-hosts is still not completely understood. Clubroot resistance assay on European clubroot differentials (ECD) set revealed that ECD10 (Brassica napus) and ECD4 (Brassica rapa) show a complete resistance to the tested P. brassicae isolate in contrast to highly susceptible hosts Westar (B. napus) and ECD5 (B. rapa). Previously, we used fluorescent probe-based confocal microscopy (FCM) to refine the life cycle of P. brassicae and indicate the important time points during its infection in Arabidopsis. Here, we used FCM to systematically investigate the infection of P. brassicae in two resistant host species ECD10 and ECD4 and two non-host crops wheat and barley at each indicated time points, compared with two susceptible hosts Westar and ECD5. We found that P. brassicae can initiate the primary infection phase and produce uninucleate primary plasmodia in both resistant hosts and non-hosts just like susceptible hosts at 2 days post-inoculation (dpi). Importantly, P. brassicae can develop into zoosporangia and secondary zoospores and release the secondary zoospores from the zoosporangia in resistant hosts at 7 dpi, comparable to susceptible hosts. However, during the secondary infection phase, no secondary plasmodium was detected in the cortical cells of both resistant hosts in contrast to massive secondary plasmodia present in the cortex tissue of two susceptible hosts leading to root swelling at 15 dpi. In both non-host crops, only uninucleate primary plasmodia were observed throughout roots at 7 and 15 dpi. Quantitative PCR based on DNA revealed that the biomass of P. brassicae has no significant increase from 2 dpi in non-host plants and from 7 dpi in resistant host plants, compared to the huge biomass increase in susceptible host plants from 2 to 25 dpi. Our study reveals that the primary infection phase in the root epidermis and the secondary infection phase in the cortex tissue are, respectively, blocked in non-hosts and resistant hosts, contributing to understanding of cellular and molecular mechanisms underlying clubroot non-host and host resistance.

Receptor-Like Kinases BAK1 and SOBIR1 Are Required for Necrotizing Activity of a Novel Group of Sclerotinia sclerotiorum Necrosis-Inducing Effectors.

Sclerotinia sclerotiorum is a characteristic necrotrophic plant pathogen and is dependent on the induction of host cell death for nutrient acquisition. To identify necrosis-inducing effectors, the genome of S. sclerotiorum was scanned for genes encoding small, secreted, cysteine-rich proteins. These potential effectors were tested for their ability to induce necrosis in Nicotiana benthamiana via Agrobacterium-mediated expression and for cellular localization in host cells. Six novel proteins were discovered, of which all but one required a signal peptide for export to the apoplast for necrotizing activity. Virus-induced gene silencing revealed that the five necrosis-inducing effectors with a requirement for secretion also required the plant co-receptor-like kinases Brassinosteroid Insensitive 1-Associated Receptor Kinase 1 (BAK1) and Suppressor of BAK1-Interacting Receptor-like Kinase 1 (SOBIR1) for the induction of necrosis. S. sclerotiorum necrosis-inducing effector 2 (SsNE2) represented a new class of necrosis-inducing proteins as orthologs were identified in several other phytopathogenic fungi that were also capable of inducing necrosis. Substitution of conserved cysteine residues with alanine reduced, but did not abolish, the necrotizing activity of SsNE2 and full-length protein was required for function as peptides spanning the entire protein were unable to induce necrosis. These results illustrate the importance of necrosis-inducing effectors for S. sclerotiorum virulence and the role of host extracellular receptor(s) in effector-triggered susceptibility to this pathogen.

Arabidopsis UBC22, an E2 able to catalyze lysine-11 specific ubiquitin linkage formation, has multiple functions in plant growth and immunity.

Protein ubiquitination is critical for various biological processes in eukaryotes. A ubiquitin (Ub) chain can be linked through one of the seven lysine (K) residues or the N-terminus methionine of the Ub, and the Ub-conjugating enzymes called E2s play a critical role in determining the linkage specificity of Ub chains. Further, while K48-linked polyubiquitin chain is important for protein degradation, much less is known about the functions of other types of polyubiquitin chains in plants. We showed previously that UBC22 is unique in its ability to catalyze K11-dependent Ub dimer formation in vitro and ubc22 knockout mutants had defects in megasporogenesis. In this study, further analyses of the Arabidopsis ubc22 mutants revealed four subtypes of plants based on the phenotypic changes in vegetative growth. These four subtypes appeared consistently in the plants of three independent ubc22 mutants. Transcriptomic analysis showed that transcript levels of genes related to several pathways were altered differently in different subtypes of mutant plants. In one subtype, the mutant plants had increased expression of genes related to plant defenses and showed enhanced resistance to a necrotrophic plant pathogen. These results suggest multiple functions of UBC22 during plant development and stress response.

Refining the Life Cycle of Plasmodiophora brassicae.

As a soilborne protist pathogen, Plasmodiophora brassicae causes the devastating clubroot disease on Brassicaeae crops worldwide. Due to its intracellular obligate biotrophic nature, the life cycle of P. brassicae is still not fully understood. Here, we used fluorescent probe-based confocal microscopy and transmission electron microscopy (TEM) to investigate the infection process of P. brassicae on the susceptible host Arabidopsis under controlled conditions. We found that P. brassicae can initiate the primary infection in both root hairs and epidermal cells, producing the uninucleate primary plasmodium at 1 day postinoculation (dpi). After that, the developed multinucleate primary plasmodium underwent condensing and cytoplasm cleavage into uninucleate zoosporangia from 1 to 4 dpi. This was subsequently followed by the formation of multinucleate zoosporangia and the production of secondary zoospores within zoosporangium. Importantly, the secondary zoospores performed a conjugation in the root epidermal cells after their release. TEM revealed extensive uninucleate secondary plasmodium in cortical cells at 8 dpi, indicating the establishment of the secondary infection. The P. brassicae subsequently developed into binucleate, quadrinucleate, and multinucleate secondary plasmodia from 10 to 15 dpi, during which the clubroot symptoms appeared. The uninucleate resting spores were first observed in the cortical cells at 24 dpi, marking the completion of a life cycle. We also provided evidence that the secondary infection of P. brassicae may represent the diploid sexual life stage. From these findings, we propose a refined life cycle of P. brassicae which will contribute to understanding of the complicated infection biology of P. brassicae.

Specific Recruitment of Phosphoinositide Species to the Plant-Pathogen Interfacial Membrane Underlies Arabidopsis Susceptibility to Fungal Infection.

Different phosphoinositides enriched at the membranes of specific subcellular compartments within plant cells contribute to organelle identity, ensuring appropriate cellular trafficking and function. During the infection of plant cells, biotrophic pathogens such as powdery mildews enter plant cells and differentiate into haustoria. Each haustorium is enveloped by an extrahaustorial membrane (EHM) derived from the host plasma membrane. Little is known about the EHM biogenesis and identity. Here, we demonstrate that among the two plasma membrane phosphoinositides in Arabidopsis (Arabidopsis thaliana), PI(4,5)P2 is dynamically up-regulated at powdery mildew infection sites and recruited to the EHM, whereas PI4P is absent in the EHM. Lateral transport of PI(4,5)P2 into the EHM occurs through a brefeldin A-insensitive but actin-dependent trafficking pathway. Furthermore, the lower levels of PI(4,5)P2 in pip5k1 pip5k2 mutants inhibit fungal pathogen development and cause disease resistance, independent of cell death-associated defenses and involving impaired host susceptibility. Our results reveal that plant biotrophic and hemibiotrophic pathogens modulate the subcellular distribution of host phosphoinositides and recruit PI(4,5)P2 as a susceptibility factor for plant disease.