Conserved oligomeric Golgi (COG) complex genes functioning in defense are expressed in root cells undergoing a defense response to a pathogenic infection and exhibit regulation my MAPKs.

The conserved oligomeric Golgi (COG) complex maintains correct Golgi structure and function during retrograde trafficking. Glycine max has 2 paralogs of each COG gene, with one paralog of each gene family having a defense function to the parasitic nematode Heterodera glycines. Experiments presented here show G. max COG paralogs functioning in defense are expressed specifically in the root cells (syncytia) undergoing the defense response. The expressed defense COG gene COG7-2-b is an alternate splice variant, indicating specific COG variants are important to defense. Transcriptomic experiments examining RNA isolated from COG overexpressing and RNAi roots show some COG genes co-regulate the expression of other COG complex genes. Examining signaling events responsible for COG expression, transcriptomic experiments probing MAPK overexpressing roots show their expression influences the relative transcript abundance of COG genes as compared to controls. COG complex paralogs are shown to be found in plants that are agriculturally relevant on a world-wide scale including Manihot esculenta, Zea mays, Oryza sativa, Triticum aestivum, Hordeum vulgare, Sorghum bicolor, Brassica rapa, Elaes guineensis and Saccharum officinalis and in additional crops significant to U.S. agriculture including Beta vulgaris, Solanum tuberosum, Solanum lycopersicum and Gossypium hirsutum. The analyses provide basic information on COG complex biology, including the coregulation of some COG genes and that MAPKs functioning in defense influence their expression. Furthermore, it appears in G. max and likely other crops that some level of neofunctionalization of the duplicated genes is occurring. The analysis has identified important avenues for future research broadly in plants.

Bph30 confers resistance to brown planthopper by fortifying sclerenchyma in rice leaf sheaths.

Phloem-feeding insects cause massive losses in agriculture and horticulture. Host plant resistance to phloem-feeding insects is often mediated by changes in phloem composition, which deter insect settling and feeding and decrease viability. Here, we report that rice plant resistance to the phloem-feeding brown planthopper (BPH) is associated with fortification of the sclerenchyma tissue, which is located just beneath the epidermis and a cell layer or two away from the vascular bundle in the rice leaf sheath. We found that BPHs prefer to feed on the smooth and soft region on the surface of rice leaf sheaths called the long-cell block. We identified Bph30 as a rice BPH resistance gene that prevents BPH stylets from reaching the phloem due to the fortified sclerenchyma. Bph30 is strongly expressed in sclerenchyma cells and enhances cellulose and hemicellulose synthesis, making the cell walls stiffer and sclerenchyma thicker. The structurally fortified sclerenchyma is a formidable barrier preventing BPH stylets from penetrating the leaf sheath tissues and arriving at the phloem to feed. Bph30 belongs to a novel gene family, encoding a protein with two leucine-rich domains. Another member of the family, Bph40, also conferred resistance to BPH. Collectively, the fortified sclerenchyma-mediated resistance mechanism revealed in this study expands our understanding of plant-insect interactions and opens a new path for controlling planthoppers in rice.

A Combinational Approach of Enhanced Methanol Production and Double Bt Genes for Broad Spectrum Insect Resistance in Transgenic Cotton.

The prevalence of insect resistance against Bt toxins has led to the idea of enhancing demethylation from cell wall pectin by pectin methylesterase enzyme for overproduction of methanol which is toxic to insects pests. The AtPME and AnPME fragments ligated into pCAMBIA1301 vector were confirmed through restriction digestion with EcoR1 and BamH1. Excision of 3363 bp fragment from 11,850 bp vector confirmed the ligation of both fragments into pCAMBIA1301 vector. Transformation of pectin methylesterase-producing genes, i.e., AtPME and AnPME from Arabidopsis thaliana and Aspergillus niger cloned in plant expression vector pCAMBIA1301 under 35S promoter into cotton variety CEMB-33 harboring two Bt genes Cry1Ac and Cry2A, respectively, was done by using shoot apex-cut Agrobacterium-mediated transformation method. The plantlets were screened on MS medium supplemented with hygromycin on initial basis. Amplification of 412 and 543 bp, respectively, through gene-specific primer has been obtained which confirmed the successful introduction of pCAMBIA AtPME and AnPME genes into cotton variety CEMB 33. Relative expression of AtPME and AnPME genes through real-time PCR determined the expression level of both gene ranges between 3- and 3.5-fold in different transgenic cotton lines along with quantity of methanol ranging from 0.8 to 0.9% of maximum while 0.5% to 0.6% of minimum but no expression was obtained in negative non-transgenic control cotton plant with least quantity of methanol, i.e., 0.1%. Almost 100% mortality was observed in insect bioassay for Helicoverpa armigera on detached leaves bioassay and 63% for Pink Bollworm (Pectinophora gossypiella) on growing transgenic cotton bolls as compared to positive control transgenic cotton with double Bt genes where mortality was found to be 82% for H. armigera and 50% for P. gossypiella while 0% in negative control non-transgenic plants.

Effectors of Phytophthora pathogens are powerful weapons for manipulating host immunity.

MAIN CONCLUSION: This article provides an overview of the interactions between Phytophthora effectors and plant immune system components, which form a cross-linked complex network that regulates plant pathogen resistance. Pathogens secrete numerous effector proteins into plants to promote infections. Several Phytophthora species (e.g., P. infestans, P. ramorum, P. sojae, P. capsici, P. cinnamomi, and P. parasitica) are notorious pathogens that are extremely damaging to susceptible plants. Analyses of genomic data revealed that Phytophthora species produce a large group of effector proteins, which are critical for pathogenesis. And, the targets and functions of many identified Phytophthora effectors have been investigated. Phytophthora effectors can affect various aspects of plant immune systems, including plant cell proteases, phytohormones, RNAs, the MAPK pathway, catalase, the ubiquitin proteasome pathway, the endoplasmic reticulum, NB-LRR proteins, and the cell membrane. Clarifying the effector-plant interactions is important for unravelling the functions of Phytophthora effectors during pathogenesis. In this article, we review the effectors identified in recent decades and provide an overview of the effector-directed regulatory network in plants following infections by Phytophthora species.

Characterization of the gut microbiota of invasive Agrilus mali Matsumara (Coleoptera: Buprestidae) using high-throughput sequencing: uncovering plant cell-wall degrading bacteria.

The genus Agrilus comprises diverse exotic and agriculturally important wood-boring insects that have evolved efficient digestive systems. Agrilus mali Matsumara, an invasive insect, is causing extensive mortality to endangered wild apple trees in Tianshan. In this study, we present an in-depth characterization of the gut microbiota of A. mali based on high-throughput sequencing of the 16S rRNA gene and report the presence of lignocellulose-degrading bacteria. Thirty-nine operational taxonomic units (OTUs) were characterized from the larval gut. OTUs represented 6 phyla, 10 classes, 16 orders, 20 families, and 20 genera. The majority of bacterial OTUs belonged to the order Enterobacteriales which was the most abundant taxa in the larval gut. Cultivable bacteria revealed 9 OTUs that all belonged to Gammaproteobacteria. Subsequently, we examined the breakdown of plant cell-wall compounds by bacterial isolates. Among the isolates, the highest efficiency was observed in Pantoea sp., which was able to synthesize four out of the six enzymes (cellulase, cellobiase, ß-xylanase, and ß-gluconase) responsible for plant-cell wall degradation. One isolate identified as Pseudomonas orientalis exhibited lignin peroxidase activity. Our study provides the first characterization of the gut microbial diversity of A. mali larvae and shows that some cultivable bacteria play a significant role in the digestive tracts of larvae by providing nutritional needs.

Host autophagy machinery is diverted to the pathogen interface to mediate focal defense responses against the Irish potato famine pathogen.

During plant cell invasion, the oomycete Phytophthora infestans remains enveloped by host-derived membranes whose functional properties are poorly understood. P. infestans secretes a myriad of effector proteins through these interfaces for plant colonization. Recently we showed that the effector protein PexRD54 reprograms host-selective autophagy by antagonising antimicrobial-autophagy receptor Joka2/NBR1 for ATG8CL binding (Dagdas et al., 2016). Here, we show that during infection, ATG8CL/Joka2 labelled defense-related autophagosomes are diverted toward the perimicrobial host membrane to restrict pathogen growth. PexRD54 also localizes to autophagosomes across the perimicrobial membrane, consistent with the view that the pathogen remodels host-microbe interface by co-opting the host autophagy machinery. Furthermore, we show that the host-pathogen interface is a hotspot for autophagosome biogenesis. Notably, overexpression of the early autophagosome biogenesis protein ATG9 enhances plant immunity. Our results implicate selective autophagy in polarized immune responses of plants and point to more complex functions for autophagy than the widely known degradative roles.

Genomic evidence for genes encoding leucine-rich repeat receptors linked to resistance against the eukaryotic extra- and intracellular Brassica napus pathogens Leptosphaeria maculans and Plasmodiophora brassicae.

Genes coding for nucleotide-binding leucine-rich repeat (LRR) receptors (NLRs) control resistance against intracellular (cell-penetrating) pathogens. However, evidence for a role of genes coding for proteins with LRR domains in resistance against extracellular (apoplastic) fungal pathogens is limited. Here, the distribution of genes coding for proteins with eLRR domains but lacking kinase domains was determined for the Brassica napus genome. Predictions of signal peptide and transmembrane regions divided these genes into 184 coding for receptor-like proteins (RLPs) and 121 coding for secreted proteins (SPs). Together with previously annotated NLRs, a total of 720 LRR genes were found. Leptosphaeria maculans-induced expression during a compatible interaction with cultivar Topas differed between RLP, SP and NLR gene families; NLR genes were induced relatively late, during the necrotrophic phase of pathogen colonization. Seven RLP, one SP and two NLR genes were found in Rlm1 and Rlm3/Rlm4/Rlm7/Rlm9 loci for resistance against L. maculans on chromosome A07 of B. napus. One NLR gene at the Rlm9 locus was positively selected, as was the RLP gene on chromosome A10 with LepR3 and Rlm2 alleles conferring resistance against L. maculans races with corresponding effectors AvrLm1 and AvrLm2, respectively. Known loci for resistance against L. maculans (extracellular hemi-biotrophic fungus), Sclerotinia sclerotiorum (necrotrophic fungus) and Plasmodiophora brassicae (intracellular, obligate biotrophic protist) were examined for presence of RLPs, SPs and NLRs in these regions. Whereas loci for resistance against P. brassicae were enriched for NLRs, no such signature was observed for the other pathogens. These findings demonstrate involvement of (i) NLR genes in resistance against the intracellular pathogen P. brassicae and a putative NLR gene in Rlm9-mediated resistance against the extracellular pathogen L. maculans.

Modulation of Host Endocycle During Plant-Biotroph Interactions.

Recent studies have revealed that several mutualistic and parasitic biotrophic microbes induce a cell cycle variant termed the endocycle in host cells to support their growth and reproduction. Endoreduplication is a process in which cells successively replicate their genomes without mitosis resulting in an increase in nuclear DNA ploidy. Depending on the interaction, endoreduplication can support biotroph colonization and feeding structure initiation/development, and/or serve as a mechanism to support enhanced metabolic demands of the microbe. When endoreduplication is inhibited in these interactions, biotroph growth or development is compromised. In this review, we summarize the molecular machinery known to mediate endocycle control in plants and highlight the role of these core components in feeding site establishment and/or nutrient acquisition for a diverse set of plant biotrophs.

Manipulation of host plant cells and tissues by gall-inducing insects and adaptive strategies used by different feeding guilds.

Biologists who study insect-induced plant galls are faced with the overwhelming diversity of plant forms and insect species. A challenge is to find common themes amidst this diversity. We discuss common themes that have emerged from our cytological and histochemical studies of diverse neotropical insect-induced galls. Gall initiation begins with recognition of reactive plant tissues by gall inducers, with subsequent feeding and/or oviposition triggering a cascade of events. Besides, to induce the gall structure insects have to synchronize their life cycle with plant host phenology. We predict that reactive oxygen species (ROS) play a role in gall induction, development and histochemical gradient formation. Controlled levels of ROS mediate the accumulation of (poly)phenols, and phytohormones (such as auxin) at gall sites, which contributes to the new cell developmental pathways and biochemical alterations that lead to gall formation. The classical idea of an insect-induced gall is a chamber lined with a nutritive tissue that is occupied by an insect that directly harvests nutrients from nutritive cells via its mouthparts, which function mechanically and/or as a delivery system for salivary secretions. By studying diverse gall-inducing insects we have discovered that insects with needle-like sucking mouthparts may also induce a nutritive tissue, whose nutrients are indirectly harvested as the gall-inducing insects feeds on adjacent vascular tissues. Activity of carbohydrate-related enzymes across diverse galls corroborates this hypothesis. Our research points to the importance of cytological and histochemical studies for elucidating mechanisms of induced susceptibility and induced resistance.

Identification and characterization of plant cell wall degrading enzymes from three glycoside hydrolase families in the cerambycid beetle Apriona japonica.

Xylophagous insects have evolved to thrive in a highly challenging environment. For example, wood-boring beetles from the family Cerambycidae feed exclusively on woody tissues, and to efficiently access the nutrients present in this sub-optimal environment, they have to cope with the lignocellulose barrier. Whereas microbes of the insect's gut flora were hypothesized to be responsible for the degradation of lignin, the beetle itself depends heavily on the secretion of a range of enzymes, known as plant cell wall degrading enzymes (PCWDEs), to efficiently digest both hemicellulose and cellulose networks. Here we sequenced the larval gut transcriptome of the Mulberry longhorn beetle, Apriona japonica (Cerambycidae, Lamiinae), in order to investigate the arsenal of putative PCWDEs secreted by this species. We combined our transcriptome with all available sequencing data derived from other cerambycid beetles in order to analyze and get insight into the evolutionary history of the corresponding gene families. Finally, we heterologously expressed and functionally characterized the A. japonica PCWDEs we identified from the transcriptome. Together with a range of endo-ß-1,4-glucanases, we describe here for the first time the presence in a species of Cerambycidae of (i) a xylanase member of the subfamily 2 of glycoside hydrolase family 5 (GH5 subfamily 2), as well as (ii) an exopolygalacturonase from family GH28. Our analyses greatly contribute to a better understanding of the digestion physiology of this important group of insects, many of which are major pests of forestry worldwide.

crw1--A novel maize mutant highly susceptible to foliar damage by the western corn rootworm beetle.

Western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is the most destructive insect pest of corn (Zea mays L.) in the United States. The adult WCR beetles derive their nourishment from multiple sources including corn pollen and silks as well as the pollen of alternate hosts. Conversely, the corn foliage is largely neglected as a food source by WCR beetles, leading to a perception of a passive interaction between the two. We report here a novel recessive mutation of corn that was identified and named after its foliar susceptibility to corn rootworm beetles (crw1). The crw1 mutant under field conditions was exceptionally susceptible to foliar damage by WCR beetles in an age-specific manner. It exhibits pleiotropic defects on cell wall biochemistry, morphology of leaf epidermal cells and lower structural integrity via differential accumulation of cell wall bound phenolic acids. These findings indicate that crw1 is perturbed in a pathway that was not previously ascribed to WCR susceptibility, as well as implying the presence of an active mechanism(s) deterring WCR beetles from devouring corn foliage. The discovery and characterization of this mutant provides a unique opportunity for genetic analysis of interactions between maize and adult WCR beetles and identify new strategies to control the spread and invasion of this destructive pest.

Nematode effector proteins: an emerging paradigm of parasitism.

Phytonematodes use a stylet and secreted effectors to modify host cells and ingest nutrients to support their growth and development. The molecular function of nematode effectors is currently the subject of intense investigation. In this review, we summarize our current understanding of nematode effectors, with a particular focus on proteinaceous stylet-secreted effectors of sedentary endoparasitic phytonematodes, for which a wealth of information has surfaced in the past 10 yr. We provide an update on the effector repertoires of several of the most economically important genera of phytonematodes and discuss current approaches to dissecting their function. Lastly, we highlight the latest breakthroughs in effector discovery that promise to shed new light on effector diversity and function across the phylum Nematoda.

Whole-mount confocal imaging of nuclei in giant feeding cells induced by root-knot nematodes in Arabidopsis.

• Excellent visualization of nuclei was obtained here using a whole-mount procedure adapted to provide high-resolution images of large, irregularly shaped nuclei. The procedure is based on tissue clearing, and fluorescent staining of nuclear DNA with the dye propidium iodide. • The method developed for standard confocal imaging was applied to large multicellular root swellings, named galls, induced in plant hosts by the root-knot nematode Meloidogyne incognita. • Here, we performed a functional analysis, and examined the nuclear structure in giant feeding cells overexpressing the cell cycle inhibitor Kip-related protein 4 (KRP4). Ectopic KRP4 expression in galls led to aberrant nuclear structure, disturbing giant cell expansion and nematode reproduction. In vivo live-cell imaging of GFP-KRP4 demonstrated that this protein co-localizes to chromosomes from prophase to late anaphase during cell cycle progression. • The data presented here suggest the involvement of KRP4 during mitotic progression in plant cells. The detailed results obtained using confocal analysis also demonstrate the potential utility of a rapid, easy-to-use clearing method for the analysis of the nuclei of certain Arabidopsis mutants and other complex plant nuclei.

The Arabidopsis microRNA396-GRF1/GRF3 regulatory module acts as a developmental regulator in the reprogramming of root cells during cyst nematode infection.

The syncytium is a unique plant root organ whose differentiation is induced by plant-parasitic cyst nematodes to create a source of nourishment. Syncytium formation involves the redifferentiation and fusion of hundreds of root cells. The underlying regulatory networks that control this unique change of plant cell fate are not understood. Here, we report that a strong down-regulation of Arabidopsis (Arabidopsis thaliana) microRNA396 (miR396) in cells giving rise to the syncytium coincides with the initiation of the syncytial induction/formation phase and that specific miR396 up-regulation in the developed syncytium marks the beginning of the maintenance phase, when no new cells are incorporated into the syncytium. In addition, our results show that miR396 in fact has a role in the transition from one phase to the other. Expression modulations of miR396 and its Growth-Regulating Factor (GRF) target genes resulted in reduced syncytium size and arrested nematode development. Furthermore, genome-wide expression profiling revealed that the miR396-GRF regulatory system can alter the expression of 44% of the more than 7,000 genes reported to change expression in the Arabidopsis syncytium. Thus, miR396 represents a key regulator for the reprogramming of root cells. As such, this regulatory unit represents a powerful molecular target for the parasitic animal to modulate plant cells and force them into novel developmental pathways.

How do oomycete effectors interfere with plant life?

Oomycete genomes have yielded a large number of predicted effector proteins that collectively interfere with plant life in order to create a favourable environment for pathogen infection. Oomycetes secrete effectors that can be active in the host's extracellular environment, for example inhibiting host defence enzymes, or inside host cells where they can interfere with plant processes, in particular suppression of defence. Two classes of effectors are known to be host-translocated: the RXLRs and Crinklers. Many effectors show defence-suppressive activity that is important for pathogen virulence. A striking example is AVR3a of Phytophthora infestans that targets an ubiquitin ligase, the stabilisation of which may prevent host cell death. The quest for other effector targets and mechanisms is in full swing.