Rewiring of Aminoacyl-tRNA Synthetase Localization and Interactions in Plants With Extensive Mitochondrial tRNA Gene Loss.

The number of tRNAs encoded in plant mitochondrial genomes varies considerably. Ongoing loss of bacterial-like mitochondrial tRNA genes in many lineages necessitates the import of nuclear-encoded counterparts that share little sequence similarity. Because tRNAs are involved in highly specific molecular interactions, this replacement process raises questions about the identity and trafficking of enzymes necessary for the maturation and function of newly imported tRNAs. In particular, the aminoacyl-tRNA synthetases (aaRSs) that charge tRNAs are usually divided into distinct classes that specialize on either organellar (mitochondrial and plastid) or nuclear-encoded (cytosolic) tRNAs. Here, we investigate the evolution of aaRS subcellular localization in a plant lineage (Sileneae) that has experienced extensive and rapid mitochondrial tRNA loss. By analyzing full-length mRNA transcripts (PacBio Iso-Seq), we found predicted retargeting of many ancestrally cytosolic aaRSs to the mitochondrion and confirmed these results with colocalization microscopy assays. However, we also found cases where aaRS localization does not appear to change despite functional tRNA replacement, suggesting evolution of novel interactions and charging relationships. Therefore, the history of repeated tRNA replacement in Sileneae mitochondria reveals that differing constraints on tRNA/aaRS interactions may determine which of these alternative coevolutionary paths is used to maintain organellar translation in plant cells.

A dedicated flavin-dependent monooxygenase catalyzes the hydroxylation of demethoxyubiquinone into ubiquinone (coenzyme Q) in Arabidopsis.

Ubiquinone (Coenzyme Q) is a vital respiratory cofactor and liposoluble antioxidant. In plants, it is not known how the C-6 hydroxylation of demethoxyubiquinone, the penultimate step in ubiquinone biosynthesis, is catalyzed. The combination of cross-species gene network modeling along with mining of embryo-defective mutant databases of Arabidopsis thaliana identified the embryo lethal locus EMB2421 (At1g24340) as a top candidate for the missing plant demethoxyubiquinone hydroxylase. In marked contrast with prototypical eukaryotic demethoxyubiquinone hydroxylases, the catalytic mechanism of which depends on a carboxylate-bridged di-iron domain, At1g24340 is homologous to FAD-dependent oxidoreductases that instead use NAD(P)H as an electron donor. Complementation assays in Saccharomyces cerevisiae and Escherichia coli demonstrated that At1g24340 encodes a functional demethoxyubiquinone hydroxylase and that the enzyme displays strict specificity for the C-6 position of the benzoquinone ring. Laser-scanning confocal microscopy also showed that GFP-tagged At1g24340 is targeted to mitochondria. Silencing of At1g24340 resulted in 40 to 74% decrease in ubiquinone content and de novo ubiquinone biosynthesis. Consistent with the role of At1g24340 as a benzenoid ring modification enzyme, this metabolic blockage could not be bypassed by supplementation with 4-hydroxybenzoate, the immediate precursor of ubiquinone's ring. Unlike in yeast, in Arabidopsis overexpression of demethoxyubiquinone hydroxylase did not boost ubiquinone content. Phylogenetic reconstructions indicated that plant demethoxyubiquinone hydroxylase is most closely related to prokaryotic monooxygenases that act on halogenated aromatics and likely descends from an event of horizontal gene transfer between a green alga and a bacterium.

Modeling the impact of evolving biofilms on flow in porous media inside a microfluidic channel.

This study integrates microfluidic experiments and mathematical modeling to study the impacts of biofilms on flow in porous media and to explore approaches to simplify modeling permeability with complicated biofilm geometries. E. coli biofilms were grown in a microfluidic channel packed with a single layer of glass beads to reach three biofilm levels: low, intermediate, and high, with biofilm ratios (ßr) of 2.7%, 17.6%, and 55.2%, respectively. Two-dimensional biofilm structures and distributions in the porous medium were modeled by digitizing confocal images and considering broad ranges of biofilm permeability (kb) (from 10-15 m2 to 10-7 m2) and biofilm porosity (εb) (from 0.2 to 0.8). The overall permeability of the porous medium (k), the flow pathways and the overall/local pressure gradients were found to be highly dependent on ßr and kb but were moderately impacted by εb when the biofilm levels were high and intermediate with kb>10-11 m2. When biofilm structures are well developed, simplified biofilm geometries, such as uniform coating and symmetric contact filling, can provide reasonable approximations of k.

Impact of mixed biofilm formation with environmental microorganisms on E. coli O157:H7 survival against sanitization.

Biofilm formation by foodborne pathogens is a serious threat to food safety and public health. Meat processing plants may harbor various microorganisms and occasional foodborne pathogens; thus, the environmental microbial community might impact pathogen survival via mixed biofilm formation. We collected floor drain samples from two beef plants with different E. coli O157:H7 prevalence history and investigated the effects of the environmental microorganisms on pathogen sanitizer tolerance. The results showed that biofilm forming ability and bacterial species composition varied considerably based on the plants and drain locations. E. coli O157:H7 cells obtained significantly higher sanitizer tolerance in mixed biofilms by samples from the plant with recurrent E. coli O157:H7 prevalence than those mixed with samples from the other plant. The mixed biofilm that best protected E. coli O157:H7 also had the highest species diversity. The percentages of the species were altered significantly after sanitization, suggesting that the community composition affects the role and tolerance level of each individual species. Therefore, the unique environmental microbial community, their ability to form biofilms on contact surfaces and the interspecies interactions all play roles in E. coli O157:H7 persistence by either enhancing or reducing pathogen survival within the biofilm community.

Spermine-mediated tight sealing of the Magnaporthe oryzae appressorial pore-rice leaf surface interface.

Cellular adhesion mediates many important plant-microbe interactions. In the devastating blast fungus Magnaporthe oryzae1, powerful glycoprotein-rich mucilage adhesives2 cement melanized and pressurized dome-shaped infection cells-appressoria-to host rice leaf surfaces. Enormous internal turgor pressure is directed onto a penetration peg emerging from the unmelanized, thin-walled pore at the appressorial base1-4, forcing it through the leaf cuticle where it elongates invasive hyphae in underlying epidermal cells5. Mucilage sealing around the appressorial pore facilitates turgor build-up2, but the molecular underpinnings of mucilage secretion and appressorial adhesion are unknown. Here, we discovered an unanticipated and sole role for spermine in facilitating mucilage production by mitigating endoplasmic reticulum (ER) stress in the developing appressorium. Mutant strains lacking the spermine synthase-encoding gene SPS1 progressed through all stages of appressorial development, including penetration peg formation, but cuticle penetration was unsuccessful due to reduced appressorial adhesion, which led to solute leakage. Mechanistically, spermine neutralized off-target oxygen free radicals produced by NADPH oxidase-1 (Nox1)3,6 that otherwise elicited ER stress and the unfolded protein response, thereby critically reducing mucilage secretion. Our study reveals that spermine metabolism via redox buffering of the ER underpins appressorial adhesion and rice cell invasion and provides insights into a process that is fundamental to host plant infection.

Arabidopsis 4-COUMAROYL-COA LIGASE 8 contributes to the biosynthesis of the benzenoid ring of coenzyme Q in peroxisomes.

Plants have evolved the ability to derive the benzenoid moiety of the respiratory cofactor and antioxidant, ubiquinone (coenzyme Q), either from the ß-oxidative metabolism of p-coumarate or from the peroxidative cleavage of kaempferol. Here, isotopic feeding assays, gene co-expression analysis and reverse genetics identified Arabidopsis 4-COUMARATE-COA LIGASE 8 (4-CL8; At5g38120) as a contributor to the ß-oxidation of p-coumarate for ubiquinone biosynthesis. The enzyme is part of the same clade (V) of acyl-activating enzymes than At4g19010, a p-coumarate CoA ligase known to play a central role in the conversion of p-coumarate into 4-hydroxybenzoate. A 4-cl8 T-DNA knockout displayed a 20% decrease in ubiquinone content compared with wild-type plants, while 4-CL8 overexpression boosted ubiquinone content up to 150% of the control level. Similarly, the isotopic enrichment of ubiquinone's ring was decreased by 28% in the 4-cl8 knockout as compared with wild-type controls when Phe-[Ring-13C6] was fed to the plants. This metabolic blockage could be bypassed via the exogenous supply of 4-hydroxybenzoate, the product of p-coumarate ß-oxidation. Arabidopsis 4-CL8 displays a canonical peroxisomal targeting sequence type 1, and confocal microscopy experiments using fused fluorescent reporters demonstrated that this enzyme is imported into peroxisomes. Time course feeding assays using Phe-[Ring-13C6] in a series of Arabidopsis single and double knockouts blocked in the ß-oxidative metabolism of p-coumarate (4-cl8; at4g19010; at4g19010 × 4-cl8), flavonol biosynthesis (flavanone-3-hydroxylase), or both (at4g19010 × flavanone-3-hydroxylase) indicated that continuous high light treatments (500 µE m-2 s-1; 24 h) markedly stimulated the de novo biosynthesis of ubiquinone independently of kaempferol catabolism.

An Arabidopsis protoplast isolation method reduces cytosolic acidification and activation of the chloroplast stress sensor SENSITIVE TO FREEZING 2.

Chloroplasts adapt to freezing and other abiotic stresses in part by modifying their membranes. One key-remodeling enzyme is SENSITIVE TO FREEZING2 (SFR2). SFR2 is unusual because it does not respond to initial cold stress or cold acclimation, instead it responds during freezing conditions in Arabidopsis. This response has been shown to be sensitive to cytosolic acidification. The unique lipid products of SFR2 have also been detected in response to non-freezing stresses, but what causes SFR2 to respond in these stresses is unknown. Here, we investigate protoplast isolation as a representative of wounding stress. We show that SFR2 oligogalactolipid products accumulate during protoplast isolation. Notably, we show that protoplast cytosol is acidified during isolation. Modification of the buffers reduces oligogalactolipid accumulation, while prolonged incubation in the isolated state increases it. We conclude that SFR2 activation during protoplast isolation correlates with cytosolic acidification, implying that all SFR2 activation may be dependent on cytosolic acidification. We also conclude that protoplasts can be more gently isolated, reducing their stress.

Correction: TOR-autophagy branch signaling via Imp1 dictates plant-microbe biotrophic interface longevity.

[This corrects the article DOI: 10.1371/journal.pgen.1007814.].

Prostate tumor cell exosomes containing hyaluronidase Hyal1 stimulate prostate stromal cell motility by engagement of FAK-mediated integrin signaling.

The hyaluronidase Hyal1 is clinically and functionally implicated in prostate cancer progression and metastasis. Elevated Hyal1 accelerates vesicular trafficking in prostate tumor cells, thereby enhancing their metastatic potential in an autocrine manner through increased motility and proliferation. In this report, we found Hyal1 protein is a component of exosomes produced by prostate tumor cell lines overexpressing Hyal1. We investigated the role of exosomally shed Hyal1 in modulating tumor cell autonomous functions and in modifying the behavior of prostate stromal cells. Catalytic activity of Hyal1 was necessary for enrichment of Hyal1 in the exosome fraction, which was associated with increased presence of LC3BII, an autophagic marker, in the exosomes. Hyal1-positive exosome contents were internalized from the culture medium by WPMY-1 prostate stromal fibroblasts. Treatment of prostate stromal cells with tumor exosomes did not affect proliferation, but robustly stimulated their migration in a manner dependent on Hyal1 catalytic activity. Increased motility of exosome-treated stromal cells was accompanied by enhanced adhesion to a type IV collagen matrix, as well as increased FAK phosphorylation and integrin engagement through dynamic membrane residence of ß1 integrins. The presence of Hyal1 in tumor-derived exosomes and its ability to impact the behavior of stromal cells suggests cell-cell communication via exosomes is a novel mechanism by which elevated Hyal1 promotes prostate cancer progression.

A Plant Immune Receptor Degraded by Selective Autophagy.

Plants recycle non-activated immune receptors to maintain a functional immune system. The Arabidopsis immune receptor kinase FLAGELLIN-SENSING 2 (FLS2) recognizes bacterial flagellin. However, the molecular mechanisms by which non-activated FLS2 and other non-activated plant PRRs are recycled remain not well understood. Here, we provide evidence showing that Arabidopsis orosomucoid (ORM) proteins, which have been known to be negative regulators of sphingolipid biosynthesis, act as selective autophagy receptors to mediate the degradation of FLS2. Arabidopsis plants overexpressing ORM1 or ORM2 have undetectable or greatly diminished FLS2 accumulation, nearly lack FLS2 signaling, and are more susceptible to the bacterial pathogen Pseudomonas syringae. On the other hand, ORM1/2 RNAi plants and orm1 or orm2 mutants generated by the CRISPR/Cas9-mediated gene editing have increased FLS2 accumulation and enhanced FLS2 signaling, and are more resistant to P. syringae. ORM proteins interact with FLS2 and the autophagy-related protein ATG8. Interestingly, overexpression of ORM1 or ORM2 in autophagy-defective mutants showed FLS2 abundance that is comparable to that in wild-type plants. Moreover, FLS2 levels were not decreased in Arabidopsis plants overexpressing ORM1/2 derivatives that do not interact with ATG8. Taken together, these results suggest that selective autophagy functions in maintaining the homeostasis of a plant immune receptor and that beyond sphingolipid metabolic regulation ORM proteins can also act as selective autophagy receptors.

Application of Autofluorescence for Confocal Microscopy to Aid in Archaeoparasitological Analyses.

Confocal laser scanning microscopy (CLSM) was used to examine archaeoparasitological specimens from coprolites associated with La Cueva de los Muertos Chiquitos (CMC) located near present-day Durango, Mexico. The eggs for 4 different types of parasites recovered from CMC coprolites were imaged using CLSM to assist with identification efforts. While some of the parasite eggs recovered from CMC coprolites were readily identified using standard light microscopy (LM), CLSM provided useful data for more challenging identifications by highlighting subtle morphological features and enhancing visualization of parasite egg anatomy. While other advanced microscopy techniques, such as scanning electron microscopy (SEM), may also detect cryptic identifying characters, CLSM is less destructive to the specimens. Utilizing CLSM allows for subsequent examinations, such as molecular analyses, that cannot be performed following SEM sample preparation and imaging. Furthermore, CLSM detects intrinsic autofluorescence molecules, making improved identification independent of resource and time-intensive protocols. These aspects of CLSM make it an excellent method for assisting in taxonomic identification and for acquiring more detailed images of archaeoparasitological specimens.

TOR-autophagy branch signaling via Imp1 dictates plant-microbe biotrophic interface longevity.

Like other intracellular eukaryotic phytopathogens, the devastating rice blast fungus Magnaporthe (Pyricularia) oryzae first infects living host cells by elaborating invasive hyphae (IH) surrounded by a plant-derived membrane. This forms an extended biotrophic interface enclosing an apoplastic compartment into which fungal effectors can be deployed to evade host detection. M. oryzae also forms a focal, plant membrane-rich structure, the biotrophic interfacial complex (BIC), that accumulates cytoplasmic effectors for translocation into host cells. Molecular decision-making processes integrating fungal growth and metabolism in host cells with interface function and dynamics are unknown. Here, we report unanticipated roles for the M. oryzae Target-of-Rapamycin (TOR) nutrient-signaling pathway in mediating plant-fungal biotrophic interface membrane integrity. Through a forward genetics screen for M. oryzae mutant strains resistant to the specific TOR kinase inhibitor rapamycin, we discovered IMP1 encoding a novel vacuolar protein required for membrane trafficking, V-ATPase assembly, organelle acidification and autophagy induction. During infection, Δimp1 deletants developed intracellular IH in the first infected rice cell following cuticle penetration. However, fluorescently labeled effector probes revealed that interface membrane integrity became compromised as biotrophy progressed, abolishing the BIC and releasing apoplastic effectors into host cytoplasm. Growth between rice cells was restricted. TOR-independent autophagy activation in Δimp1 deletants (following infection) remediated interface function and cell-to-cell growth. Autophagy inhibition in wild type (following infection) recapitulated Δimp1. In addition to vacuoles, Imp1GFP localized to IH membranes in an autophagy-dependent manner. Collectively, our results suggest TOR-Imp1-autophagy branch signaling mediates membrane homeostasis to prevent catastrophic erosion of the biotrophic interface, thus facilitating fungal growth in living rice cells. The significance of this work lays in elaborating a novel molecular mechanism of infection stressing the dominance of fungal metabolism and metabolic control in sustaining long-term plant-microbe interactions. This work also has implications for understanding the enigmatic biotrophy to necrotrophy transition.

Manufacturing human pluripotent stem cell derived endothelial cells in scalable and cell-friendly microenvironments.

Human pluripotent stem cell derived endothelial cells (hPSC-ECs) are of great value for studying and treating vascular diseases. However, manufacturing high quantity and quality hPSC-ECs with current cell culture technologies remains very challenging. Here, we report a novel method that can manufacture hPSC-ECs in scalable and cell-friendly microenvironments to address this challenge. Using this method, hPSCs are expanded and differentiated into ECs in microscale alginate hydrogel tubes. The hydrogel tubes protect cells from the highly variable hydrodynamic conditions and critical hydrodynamic stresses in the culture vessel and limit the cell mass less than the diffusion limits (of human tissue) to ensure efficient mass transport. The hydrogel tubes provide uniform and friendly microenvironments for cells to grow. This novel design leads to extremely high production efficiency. We showed that hPSC-ECs could be produced in 10 days with high viability (>90%), high purity (>80%) and high yield (∼5.0 × 108 cells per mL of microspace). The yield is about 250 times that of the current-state-of-the-art. hPSC-ECs made in these hydrogel tubes had similar in vitro and in vivo functions to hPSC-ECs generated by conventional cell culture methods. This simple, scalable, efficient, defined and cost-effective technology will make hPSC-ECs broadly available and affordable for various biomedical applications.

Hydrogel-Based Bioprocess for Scalable Manufacturing of Human Pluripotent Stem Cell-Derived Neural Stem Cells.

Neural stem cells derived from human pluripotent stem cells (hPSC-NSCs) are of great value for modeling diseases, developing drugs, and treating neurological disorders. However, manufacturing high-quantity and -quality hPSC-NSCs, especially for clinical applications, remains a challenge. Here, we report a chemically defined, high-yield, and scalable bioprocess for manufacturing hPSC-NSCs. hPSCs are expanded and differentiated into NSCs in microscale tubes made with alginate hydrogels. The tubes are used to isolate cells from the hydrodynamic stresses in the culture vessel and limit the radial diameter of the cell mass to less than 400 µm to ensure efficient mass transport during the culture. The hydrogel tubes provide uniform, reproducible, and cell-friendly microspaces and microenvironments for cells. With this new technology, we showed that hPSC-NSCs could be produced in 12 days with high viability (∼95%), high purity (>90%), and high yield (∼5 × 108 cells/mL of microspace). The volumetric yield is about 250 times more than the current state-of-the-art. Whole transcriptome analysis and quantitative real-time polymerase chain reaction showed that hPSC-NSCs made by this process had a similar gene expression to hPSC-NSCs made by the conventional culture technology. The produced hPSC-NSCs could mature into both neurons and glial cells in vitro and in vivo. The process developed in this paper can be used to produce large numbers of hPSC-NSCs for various biomedical applications in the future.

A Scalable and Efficient Bioprocess for Manufacturing Human Pluripotent Stem Cell-Derived Endothelial Cells.

Endothelial cells (ECs) are of great value for cell therapy, tissue engineering, and drug discovery. Obtaining high-quantity and -quality ECs remains very challenging. Here, we report a method for the scalable manufacturing of ECs from human pluripotent stem cells (hPSCs). hPSCs are expanded and differentiated into ECs in a 3D thermoreversible PNIPAAm-PEG hydrogel. The hydrogel protects cells from hydrodynamic stresses in the culture vessel and prevents cells from excessive agglomeration, leading to high-culture efficiency including high-viability (>90%), high-purity (>80%), and high-volumetric yield (2.0 × 107 cells/mL). These ECs (i.e., 3D-ECs) had similar properties as ECs made using 2D culture systems (i.e., 2D-ECs). Genome-wide gene expression analysis showed that 3D-ECs had higher expression of genes related to vasculature development, extracellular matrix, and glycolysis, while 2D-ECs had higher expression of genes related to cell proliferation.

A totally recombinant fibrin matrix for mesenchymal stem cell culture and delivery.

Mesenchymal stem cells (MSCs) have been widely studied for tissue engineering and treating diseases in laboratories, clinical trials, and clinics. Fibrin matrices are often used to culture MSCs or increase the retention of MSCs at the injection site. However, fibrins made with the human plasma derived fibrinogen have high cost and risk of human pathogen transmission. In this article, we studied if fibrin matrices made with recombinant human fibrinogen, recombinant human thrombin, and recombinant human factor XIII could be used to culture and deliver MSCs. We systematically investigated the relationships between the fibrin matrix formulation, its nanostructure, and the behaviors of the cells in the matrix including the cell morphology, viability, and growth. We found that the fibrinogen concentration significantly affected the matrix structure and cell behaviors. We then used an optimized fibrin matrix to deliver human MSCs into mice subcutaneously. We found that the matrix could significantly enhance the retention of MSCs at the injection site. To our best knowledge, this is the first study on using fibrin matrices made with entirely recombinant proteins for culturing and delivering MSCs. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3135-3142, 2018.

Specialized Plastids Trigger Tissue-Specific Signaling for Systemic Stress Response in Plants.

Plastids comprise a complex set of organelles in plants that can undergo distinctive patterns of differentiation and redifferentiation during their lifespan. Plastids localized to the epidermis and vascular parenchyma are distinctive in size, structural features, and functions. These plastids are termed "sensory" plastids, and here we show their proteome to be distinct from chloroplasts, with specialized stress-associated features. The distinctive sensory plastid proteome in Arabidopsis (Arabidopsis thaliana) derives from spatiotemporal regulation of nuclear genes encoding plastid-targeted proteins. Perturbation caused by depletion of the sensory plastid-specific protein MutS HOMOLOG1 conditioned local, programmed changes in gene networks controlling chromatin, stress-related phytohormone, and circadian clock behavior and producing a global, systemic stress response in the plant. We posit that the sensory plastid participates in sensing environmental stress, integrating this sensory function with epigenetic and gene expression circuitry to condition heritable stress memory.

Metabolic reconstructions identify plant 3-methylglutaconyl-CoA hydratase that is crucial for branched-chain amino acid catabolism in mitochondria.

The proteinogenic branched-chain amino acids (BCAAs) leucine, isoleucine and valine are essential nutrients for mammals. In plants, BCAAs double as alternative energy sources when carbohydrates become limiting, the catabolism of BCAAs providing electrons to the respiratory chain and intermediates to the tricarboxylic acid cycle. Yet, the actual architecture of the degradation pathways of BCAAs is not well understood. In this study, gene network modeling in Arabidopsis and rice, and plant-prokaryote comparative genomics detected candidates for 3-methylglutaconyl-CoA hydratase (4.2.1.18), one of the missing plant enzymes of leucine catabolism. Alignments of these protein candidates sampled from various spermatophytes revealed non-homologous N-terminal extensions that are lacking in their bacterial counterparts, and green fluorescent protein-fusion experiments demonstrated that the Arabidopsis protein, product of gene At4g16800, is targeted to mitochondria. Recombinant At4g16800 catalyzed the dehydration of 3-hydroxymethylglutaryl-CoA into 3-methylglutaconyl-CoA, and displayed kinetic features similar to those of its prokaryotic homolog. When at4g16800 knockout plants were subjected to dark-induced carbon starvation, their rosette leaves displayed accelerated senescence as compared with control plants, and this phenotype was paralleled by a marked increase in the accumulation of free and total leucine, isoleucine and valine. The seeds of the at4g16800 mutant showed a similar accumulation of free BCAAs. These data suggest that 3-methylglutaconyl-CoA hydratase is not solely involved in the degradation of leucine, but is also a significant contributor to that of isoleucine and valine. Furthermore, evidence is shown that unlike the situation observed in Trypanosomatidae, leucine catabolism does not contribute to the formation of the terpenoid precursor mevalonate.

Wheat streak mosaic virus Coat Protein Deletion Mutants Elicit More Severe Symptoms Than Wild-Type Virus in Multiple Cereal Hosts.

Previously, we reported that coat protein (CP) of Wheat streak mosaic virus (WSMV) (genus Tritimovirus, family Potyviridae) tolerates deletion of amino acids 36 to 84 for efficient systemic infection of wheat. In this study, we demonstrated that WSMV mutants with deletion of CP amino acids 58 to 84 but not of 36 to 57 induced severe chlorotic streaks and spots, followed by acute chlorosis in wheat, maize, barley, and rye compared with mild to moderate chlorotic streaks and mosaic symptoms by wild-type virus. Deletion of CP amino acids 58 to 84 from the WSMV genome accelerated cell-to-cell movement, with increased accumulation of genomic RNAs and CP, compared with the wild-type virus. Microscopic examination of wheat tissues infected by green fluorescent protein-tagged mutants revealed that infection by mutants lacking CP amino acids 58 to 84 caused degradation of chloroplasts, resulting in acute macroscopic chlorosis. The profile of CP-specific proteins was altered in wheat infected by mutants causing acute chlorosis, compared with mutants eliciting wild-type symptoms. All deletion mutants accumulated CP-specific major protein similarly to that in wild-type virus; however, mutants that elicit acute chlorosis failed to accumulate a 31-kDa minor protein compared with wild-type virus or mutants lacking amino acids 36 to 57. Taken together, these data suggest that deletion of CP amino acids 58 to 84 from the WSMV genome enhanced accumulation of CP and genomic RNA, altered CP-specific protein profiles, and caused severe symptom phenotypes in multiple cereal hosts.

The Magnaporthe oryzae nitrooxidative stress response suppresses rice innate immunity during blast disease.

Understanding how microorganisms manipulate plant innate immunity and colonize host cells is a major goal of plant pathology. Here, we report that the fungal nitrooxidative stress response suppresses host defences to facilitate the growth and development of the important rice pathogen Magnaporthe oryzae in leaf cells. Nitronate monooxygenases encoded by NMO genes catalyse the oxidative denitrification of nitroalkanes. We show that the M. oryzae NMO2 gene is required for mitigating damaging lipid nitration under nitrooxidative stress conditions and, consequently, for using nitrate and nitrite as nitrogen sources. On plants, the Δnmo2 mutant strain penetrated host cuticles like wild type, but invasive hyphal growth in rice cells was restricted and elicited plant immune responses that included the formation of cellular deposits and a host reactive oxygen species burst. Development of the M. oryzae effector-secreting biotrophic interfacial complex (BIC) was misregulated in the Δnmo2 mutant. Inhibiting or quenching host reactive oxygen species suppressed rice innate immune responses and allowed the Δnmo2 mutant to grow and develop normally in infected cells. NMO2 is thus essential for mitigating nitrooxidative cellular damage and, in rice cells, maintaining redox balance to avoid triggering plant defences that impact M. oryzae growth and BIC development.