Developing reverse genetics systems of northern cereal mosaic virus to reveal superinfection exclusion of two cytorhabdoviruses in barley plants

Recently, reverse genetics systems of plant negative‐stranded RNA (NSR) viruses have been developed to study virus–host interactions. Nonetheless, genetic rescue of plant NSR viruses in both insect vectors and monocot plants is very limited. Northern cereal mosaic virus (NCMV), a plant cytorhabdovirus, causes severe diseases in cereal plants through transmission by the small brown planthopper (SBPH, Laodelphax striatellus) in a propagative manner. In this study, we first developed a minireplicon system of NCMV in Nicotiana benthamiana plants, and then recovered a recombinant NCMV virus (rNCMV‐RFP), with a red fluorescent protein (RFP) insertion, in SBPHs and barley plants. We further used rNCMV‐RFP and green fluorescent protein (GFP)‐tagged barley yellow striate mosaic virus (rBYSMV‐GFP), a closely related cytorhabdovirus, to study superinfection exclusion, a widely observed phenomenon in dicot plants rarely studied in monocot plants. Interestingly, cellular superinfection exclusion of rBYSMV‐GFP and rNCMV‐RFP was observed in barley leaves. Our results demonstrate that two insect‐transmitted cytorhabdoviruses are enemies rather than friends at the cellular level during coinfections in plants.     

Medicarpin confers powdery mildew resistance in Medicago truncatula and activates the salicylic acid signalling pathway

Powdery mildew (PM) caused by the obligate biotrophic fungal pathogen Erysiphe pisi is an economically important disease of legumes. Legumes are rich in isoflavonoids, a class of secondary metabolites whose role in PM resistance is ambiguous. Here we show that the pterocarpan medicarpin accumulates at fungal infection sites, as analysed by fluorescein‐tagged medicarpin, and provides penetration and post‐penetration resistance against E. pisi in Medicago truncatula in part through the activation of the salicylic acid (SA) signalling pathway. Comparative gene expression and metabolite analyses revealed an early induction of isoflavonoid biosynthesis and accumulation of the defence phytohormones SA and jasmonic acid (JA) in the highly resistant M. truncatula genotype A17 but not in moderately susceptible R108 in response to PM infection. Pretreatment of R108 leaves with medicarpin increased SA levels, SA‐associated gene expression, and accumulation of hydrogen peroxide at PM infection sites, and reduced fungal penetration and colony formation. Strong parallels in the levels of medicarpin and SA, but not JA, were observed on medicarpin/SA treatment pre‐ or post‐PM infection. Collectively, our results suggest that medicarpin and SA may act in concert to restrict E. pisi growth, providing new insights into the metabolic and signalling pathways required for PM resistance in legumes.     

Coinfection with a virus constrains within‐host infection load but increases transmission potential of a highly virulent fungal plant pathogen

The trade‐off between within‐host infection rate and transmission to new hosts is predicted to constrain pathogen evolution, and to maintain polymorphism in pathogen populations. Pathogen life‐history stages and their correlations that underpin infection development may change under coinfection with other parasites as they compete for the same limited host resources. Cross‐kingdom interactions are common among pathogens in both natural and cultivated systems, yet their impacts on disease ecology and evolution are rarely studied. The host plant Plantago lanceolata is naturally infected by both Phomopsis subordinaria, a seed killing fungus, as well as Plantago lanceolata latent virus (PlLV) in the Åland Islands, SW Finland. We performed an inoculation assay to test whether coinfection with PlLV affects performance of two P. subordinaria strains, and the correlation between within‐host infection rate and transmission potential. The strains differed in the measured life‐history traits and their correlations. Moreover, we found that under virus coinfection, within‐host infection rate of P. subordinaria was smaller but transmission potential was higher compared to strains under single infection. The negative correlation between within‐host infection rate and transmission potential detected under single infection became positive under coinfection with PlLV. To understand whether within‐host and between‐host dynamics are correlated in wild populations, we surveyed 260 natural populations of P. lanceolata for P. subordinaria infection occurrence. When infections were found, we estimated between‐hosts dynamics by determining pathogen population size as the proportion of infected individuals, and within‐host dynamics by counting the proportion of infected flower stalks in 10 infected plants. In wild populations, the proportion of infected flower stalks was positively associated with pathogen population size. Jointly, our results suggest that the trade‐off between within‐host infection load and transmission may be strain specific, and that the pathogen life‐history that underpin epidemics may change depending on the diversity of infection, generating variation in disease dynamics.     

Two independent approaches converge to the cloning of a new Leptosphaeria maculans avirulence effector gene,AvrLmS‐Lep2

Brassica napus (oilseed rape, canola) seedling resistance to Leptosphaeria maculans, the causal agent of blackleg (stem canker) disease, follows a gene‐for‐gene relationship. The avirulence genes AvrLmS and AvrLep2 were described to be perceived by the resistance genes RlmS and LepR2, respectively, present in B. napus ‘Surpass 400’. Here we report cloning of AvrLmS and AvrLep2 using two independent methods. AvrLmS was cloned using combined in vitro crossing between avirulent and virulent isolates with sequencing of DNA bulks from avirulent or virulent progeny (bulked segregant sequencing). AvrLep2 was cloned using a biparental cross of avirulent and virulent L. maculans isolates and a classical map‐based cloning approach. Taking these two approaches independently, we found that AvrLmS and AvrLep2 are the same gene. Complementation of virulent isolates with this gene confirmed its role in inducing resistance on Surpass 400, Topas‐LepR2, and an RlmS‐line. The gene, renamed AvrLmS‐Lep2, encodes a small cysteine‐rich protein of unknown function with an N‐terminal secretory signal peptide, which is a common feature of the majority of effectors from extracellular fungal plant pathogens. The AvrLmS‐Lep2/LepR2 interaction phenotype was found to vary from a typical hypersensitive response through intermediate resistance sometimes towards susceptibility, depending on the inoculation conditions. AvrLmS‐Lep2 was nevertheless sufficient to significantly slow the systemic growth of the pathogen and reduce the stem lesion size on plant genotypes with LepR2, indicating the potential efficiency of this resistance to control the disease in the field.     

A jacalin‐like lectin domain‐containing protein of Sclerospora graminicola acts as an apoplastic virulence effector in plant–oomycete interactions

The plant extracellular space, including the apoplast and plasma membrane, is the initial site of plant–pathogen interactions. Pathogens deliver numerous secreted proteins, called effectors, into this region to suppress plant immunity and establish infection. Downy mildew caused by the oomycete pathogen Sclerospora graminicola (Sg) is an economically important disease of Poaceae crops including foxtail millet (Setaria italica). We previously reported the genome sequence of Sg and showed that the jacalin‐related lectin (JRL) gene family has significantly expanded in this lineage. However, the biological functions of JRL proteins remained unknown. Here, we show that JRL from Sg (SgJRL) functions as an apoplastic virulence effector. We identified eight SgJRLs by protein mass spectrometry analysis of extracellular fluid from Sg‐inoculated foxtail millet leaves. SgJRLs consist of a jacalin‐like lectin domain and an N‐terminal putative secretion signal; SgJRL expression is induced by Sg infection. Heterologous expression of three SgJRLs with N‐terminal secretion signal peptides in Nicotiana benthamiana enhanced the virulence of the pathogen Phytophthora palmivora inoculated onto the same leaves. Of the three SgJRLs, SG06536 fused with green fluorescent protein (GFP) localized to the apoplastic space in N. benthamiana leaves. INF1‐mediated induction of defence‐related genes was suppressed by co‐expression of SG06536‐GFP. These findings suggest that JRLs are novel apoplastic effectors that contribute to pathogenicity by suppressing plant defence responses.     

Cyclic di‐GMP modulates sessile‐motile phenotypes and virulence in Dickeya oryzae via two PilZ domain receptors

Dickeya oryzae is a bacterial pathogen causing the severe rice stem rot disease in China and other rice‐growing countries. We showed recently that the universal bacterial second messenger c‐di‐GMP plays an important role in modulation of bacterial motility and pathogenicity, but the mechanism of regulation remains unknown. In this study, bioinformatics analysis of the D. oryzae EC1 genome led to the identification of two proteins, YcgR and BcsA, both of which contain a conserved c‐di‐GMP receptor domain, known as the PilZ‐domain. By deleting all the genes encoding c‐di‐GMP‐degrading enzymes in D. oryzae EC1, the resultant mutant 7ΔPDE with high c‐di‐GMP levels became nonmotile, formed hyperbiofilm, and lost the ability to colonize and invade rice seeds. These phenotypes were partially reversed by deletion of ycgR in the mutant 7ΔPDE, whereas deletion of bcsA only reversed the hyperbiofilm phenotype of mutant 7ΔPDE. Significantly, double deletion of ycgR and bcsA in mutant 7ΔPDE rescued its motility, biofilm formation, and virulence to levels of wild‐type EC1. In vitro biochemical experiments and in vivo phenotypic assays further validated that YcgR and BcsA proteins are the receptors for c‐di‐GMP, which together play a critical role in regulating the c‐di‐GMP‐associated functionality. The findings from this study fill a gap in our understanding of how c‐di‐GMP modulates bacterial motility and biofilm formation, and provide useful clues for further elucidation of sophisticated virulence regulatory mechanisms in this important plant pathogen.     

Phytophthora infestans RXLR Effector PexRD2 Interacts with Host MAPKKKε to Suppress Plant Immune Signaling

Mitogen-activated protein kinase cascades are key players in plant immune signaling pathways, transducing the perception of invading pathogens into effective defense responses. Plant pathogenic oomycetes, such as the Irish potato famine pathogen Phytophthora infestans, deliver RXLR effector proteins to plant cells to modulate host immune signaling and promote colonization. Our understanding of the molecular mechanisms by which these effectors act in plant cells is limited. Here, we report that the P. infestans RXLR effector PexRD2 interacts with the kinase domain of MAPKKKε, a positive regulator of cell death associated with plant immunity. Expression of PexRD2 or silencing MAPKKKε in Nicotiana benthamiana enhances susceptibility to P. infestans. We show that PexRD2 perturbs signaling pathways triggered by or dependent on MAPKKKε. By contrast, homologs of PexRD2 from P. infestans had reduced or no interaction with MAPKKKε and did not promote disease susceptibility. Structure-led mutagenesis identified PexRD2 variants that do not interact with MAPKKKε and fail to support enhanced pathogen growth or perturb MAPKKKε signaling pathways. Our findings provide evidence that P. infestans RXLR effector PexRD2 has evolved to interact with a specific host MAPKKK to perturb plant immunity–related signaling.     

Sclerotinia sclerotiorum Thioredoxin1 (SsTrx1) is required for pathogenicity and oxidative stress tolerance

Sclerotinia sclerotiorum infects host plant tissues by inducing necrosis to source nutrients needed for its establishment. Tissue necrosis results from an enhanced generation of reactive oxygen species (ROS) at the site of infection and apoptosis. Pathogens have evolved ROS scavenging mechanisms to withstand host‐induced oxidative damage. However, the genes associated with ROS scavenging pathways are yet to be fully investigated in S. sclerotiorum. We selected the S. sclerotiorum Thioredoxin1 gene (SsTrx1) for our investigations as its expression is significantly induced during S. sclerotiorum infection. RNA interference‐induced silencing of SsTrx1 in S. sclerotiorum affected the hyphal growth rate, mycelial morphology, and sclerotial development under in vitro conditions. These outcomes confirmed the involvement of SsTrx1 in promoting pathogenicity and oxidative stress tolerance of S. sclerotiorum. We next constructed an SsTrx1‐based host‐induced gene silencing (HIGS) vector and mobilized it into Arabidopsis thaliana (HIGS‐A) and Nicotiana benthamiana (HIGS‐N). The disease resistance analysis revealed significantly reduced pathogenicity and disease progression in the transformed genotypes as compared to the nontransformed and empty vector controls. The relative gene expression of SsTrx1 increased under oxidative stress. Taken together, our results show that normal expression of SsTrx1 is crucial for pathogenicity and oxidative stress tolerance of S. sclerotiorum.     

Immune function differences between two color morphs of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae) at different life stages

Several studies demonstrated that in insects cuticle melanism is interrelated with pathogen resistance, as melanin‐based coloration and innate immunity possess similar physiological pathways. For some insects, higher pathogen resistance was observed in darker individuals than in individuals with lighter cuticular coloration. Here, we investigated the difference in immune response between two color morphs (black and red) and between the life stages (pupa and adult) of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae). Here in this study, cuticle thickness, microbial test (antimicrobial activity, phenoloxidase activity, and hemocyte density), and immune‐related gene expression were evaluated at different stages of RPW. Study results revealed that cuticle thickness of black phenotype was thicker than red phenotype at old‐pupa stage, while no significant difference found at adult stage. These results may relate to the development processes of epidermis in different stages of RPW. The results of antimicrobial activity, phenoloxidase (PO) activity, and hemocyte density analyses showed that adults with a red phenotype had stronger pathogen resistance than those with a black phenotype. In addition to antimicrobial activity and PO activity, we tested relative gene expression in the fat body of old pupae. The results of hemolymph antimicrobial analysis showed that old pupae with a red phenotype were significantly different from those with a black phenotype at 12 hr after Staphylococcus aureus injection, suggesting that red phenotype pupae were more sensitive to S. aureus. Examination of gene expression in the fat body also revealed that the red phenotype had a higher immune response than the black phenotype. Our results were inconsistent with the previous conclusion that dark insects had increased immune function, suggesting that the relationship between cuticle pigmentation and immune function in insects was not a direct link. Additional possible factors that are associated with the immune response, such as life‐history, developmental, physiological factors also need to be considered.     

Genome‐wide transcriptome signatures of ant‐farmed Squamellaria epiphytes reveal key functions in a unique symbiosis

Farming of fungi by ants, termites, or beetles has led to ecologically successful societies fueled by industrial‐scale food production. Another type of obligate insect agriculture in Fiji involves the symbiosis between the ant Philidris nagasau and epiphytes in the genus Squamellaria (Rubiaceae) that the ants fertilize, defend, harvest, and depend on for nesting. All farmed Squamellaria form tubers (domatia) with preformed entrance holes and complex cavity networks occupied by P. nagasau. The inner surface of the domatia consists of smooth‐surfaced walls where the ants nest and rear their brood, and warty‐surfaced walls where they fertilize their crop by defecation. Here, we use RNA sequencing to identify gene expression patterns associated with the smooth versus warty wall types. Since wall differentiation occurred in the most recent common ancestor of all farmed species of Squamellaria, our study also identifies genetic pathways co‐opted following the emergence of agriculture. Warty‐surfaced walls show many upregulated genes linked to auxin transport, root development, and nitrogen transport consistent with their root‐like function; their defense‐related genes are also upregulated, probably to protect these permeable areas from pathogen entry. In smooth‐surfaced walls, genes functioning in suberin and wax biosynthesis are upregulated, contributing to the formation of an impermeable ant‐nesting area in the domatium. This study throws light on a number of functional characteristics of plant farming by ants and illustrates the power of genomic studies of symbiosis.     

Partitioning of beta‐diversity reveals distinct assembly mechanisms of plant and soil microbial communities in response to nitrogen enrichment

Nitrogen (N) deposition poses a serious threat to terrestrial biodiversity and alters plant and soil microbial community composition. Species turnover and nestedness reflect the underlying mechanisms of variations in community composition. However, it remains unclear how species turnover and nestedness contribute to different responses of taxonomic groups (plants and soil microbes) to N enrichment. Here, based on a 13‐year consecutive multi‐level N addition experiment in a semiarid steppe, we partitioned community β‐diversity into species turnover and nestedness components and explored how and why plant and microbial communities reorganize via these two processes following N enrichment. We found that plant, soil bacterial, and fungal β‐diversity increased, but their two components showed different patterns with increasing N input. Plant β‐diversity was mainly driven by species turnover under lower N input but by nestedness under higher N input, which may be due to a reduction in forb species, with low tolerance to soil Mn²⁺, with increasing N input. However, turnover was the main contributor to differences in soil bacterial and fungal communities with increasing N input, indicating the phenomenon of microbial taxa replacement. The turnover of bacteria increased greatly whereas that of fungi remained within a narrow range with increasing N input. We further found that the increased soil Mn²⁺ concentration was the best predictor for increasing nestedness of plant communities under higher N input, whereas increasing N availability and acidification together contributed to the turnover of bacterial communities. However, environmental factors could explain neither fungal turnover nor nestedness. Our findings reflect two different pathways of community changes in plants, soil bacteria, and fungi, as well as their distinct community assembly in response to N enrichment. Disentangling the turnover and nestedness of plant and microbial β‐diversity would have important implications for understanding plant–soil microbe interactions and seeking conservation strategies for maintaining regional diversity.     

Sugar beet and barley yields in relation to inoculation with N2-fixing and phosphate solubilizing bacteria

Recently, there has been a resurgence of interest in bioorganic fertilizers as part of sustainable agricultural practices to alleviate drawbacks of intensive farming practices. N₂-fixing and P-solubilizing bacteria are important in plant nutrition increasing N and P uptake by the plants, and playing a significant role as plant growth-promoting rhizobacteria in the biofertilization of crops. A study was conducted in order to investigate the effects of two N₂-fixing (OSU-140 and OSU-142) and a strain of P-solubilizing bacteria (M-13) in single, dual and three strains combinations on sugar beet and barley yields under field conditions in 2001 and 2002. The treatments included: (1) Control (no inoculation and fertilizer), (2) Bacillus OSU-140, (3) Bacillus OSU-142, (4) Bacillus M-13, (5) OSU-140 + OSU-142, (6) OSU-140 + M-13, (7) OSU-142 + M-13, (8) OSU-140 + OSU-142 + M-13, (9) N, (10) NP. N and NP plots were fertilized with 120 kg N ha^–1 and 120 kg N ha^–1 + 90 kg P ha^- for sugar beet and 80 kg N ha^–1 and 80 kg N ha^–1 + 60 kg P ha^–1 for barley. The experiments were conducted in a randomized block design with five replicates. All inoculations and fertilizer applications significantly increased leaf, root and sugar yield of sugar beet and grain and biomass yields of barley over the control. Single inoculations with N₂-fixing bacteria increased sugar beet root and barley yields by 5.6–11.0% depending on the species while P-solubilizing bacteria alone gave yield increases by 5.5–7.5% compared to control. Dual inoculation and mixture of three bacteria gave increases by 7.7–12.7% over control as compared with 20.7–25.9% yield increases by NP application. Mixture of all three strains, dual inoculation of N₂-fixing OSU-142 and P-solubilizing M-13, and/or dual inoculation N₂-fixing bacteria significantly increased root and sugar yields of sugar beet, compared with single inoculations with OSU-140 or M-13. Dual inoculation of N₂-fixing Bacillus OSU-140 and OSU-142, and/or mixed inoculations with three bacteria significantly increased grain yield of barley compared with single inoculations of OSU-142 and M-13. In contrast with other combinations, dual inoculation of N₂-fixing OSU-140 and P-solubilizing M-13 did not always significantly increase leaf, root and sugar yield of sugar beet, grain and biomass yield of barley compared to single applications both with N₂-fixing bacteria. The beneficial effects of the bacteria on plant growth varied significantly depending on environmental conditions, bacterial strains, and plant and soil conditions.     

TLR13 contributes to skeletal muscle atrophy by increasing insulin resistance in chronic kidney disease

ObjectivesInsulin resistance in chronic kidney disease (CKD) stimulates muscle wasting, but the molecular processes behind the resistance are undetermined. However, inflammation in skeletal muscle is implicated in the pathogenesis of insulin resistance and cachexia. Toll‐like receptors (TLRs) are known to regulate local innate immune responses, and microarray data have shown that Tlr13 is upregulated in the muscles of mice with CKD, but the relevance is unknown.Materials and MethodsWe performed in vitro experiments in C2C12 myotubes and constructed a CKD murine model using subtotal nephrectomy to conduct experiments in vivo.Results Tlr13 expression was stimulated in C2C12 myotubes treated with uremic serum. The expression of Tlr13 was also upregulated in the tibialis anterior muscles of mice with CKD. Tlr13 knockdown with siRNAs in skeletal muscle cells decreased insulin resistance despite the inclusion of uremic serum. This led to increased levels of p‐AKT and suppression of protein degradation. Using immunofluorescence staining and coimmunoprecipitation assay, we found that TLR13 recruits IRF3, which activates Irf3 expression, resulting in decreased AKT activity. Moreover, insulin resistance and proteolysis are re‐induced by Irf3 overexpression under Tlr13 deletion.ConclusionsOur results indicate that TLR13 is involved in CKD‐mediated insulin resistance in muscle. In catabolic conditions where insulin signaling is impaired, targeting TLR13 may improve insulin sensitivity and prevent muscle atrophy.     

Engineering a genome‐reduced bacterium to eliminate Staphylococcus aureus biofilms in vivo

Bacteria present a promising delivery system for treating human diseases. Here, we engineered the genome‐reduced human lung pathogen Mycoplasma pneumoniae as a live biotherapeutic to treat biofilm‐associated bacterial infections. This strain has a unique genetic code, which hinders gene transfer to most other bacterial genera, and it lacks a cell wall, which allows it to express proteins that target peptidoglycans of pathogenic bacteria. We first determined that removal of the pathogenic factors fully attenuated the chassis strain in vivo. We then designed synthetic promoters and identified an endogenous peptide signal sequence that, when fused to heterologous proteins, promotes efficient secretion. Based on this, we equipped the chassis strain with a genetic platform designed to secrete antibiofilm and bactericidal enzymes, resulting in a strain capable of dissolving Staphylococcus aureus biofilms preformed on catheters in vitro, ex vivo, and in vivo. To our knowledge, this is the first engineered genome‐reduced bacterium that can fight against clinically relevant biofilm‐associated bacterial infections.