Evidence for a Role of the Regulator of G-Protein Signaling Protein CPRGS-1 in Gα Subunit CPG-1-Mediated Regulation of Fungal Virulence, Conidiation, and Hydrophobin Synthesis in the Chestnut Blight Fungus Cryphonectria parasitica

We previously reported that the chestnut blight fungus Cryphonectria parasitica expresses at least three G-protein α subunits and that Gα subunit CPG-1 is essential for regulated growth, pigmentation, sporulation, and virulence. We now report the cloning and characterization of a C. parasitica regulator of G-protein signaling (RGS) protein, CPRGS-1. The phylogenetic relationship of CPRGS-1 to orthologs from other fungi was inferred and found to be generally concordant with species relationships based on 18S ribosomal sequences and on morphology. However, Hemiascomycotine RGS branch lengths in particular were longer than for their 18S sequence counterparts, which correlates with functional diversification in the signaling pathway. Deletion of cprgs-1 resulted in reduced growth, sparse aerial mycelium, and loss of pigmentation, sporulation, and virulence. Disruption of cprgs-1 was also accompanied by a severe posttranscriptional reduction in accumulation of CPG-1 and Gβ subunit CPGB-1 and severely reduced expression of the hydrophobin-encoding gene cryparin. The changes in phenotype, cryparin expression, and CPGB-1 accumulation resulting from cprgs-1 gene deletion were also observed in a strain containing a mutationally activated copy of CPG-1 but not in strains containing constitutively activated mutant alleles of the other two identified Gα subunits, CPG-2 and CPG-3. Furthermore, cprgs-1 transcript levels were increased in the activated CPG-1 strain but were unaltered in activated CPG-2 and CPG-3 strains. The results strongly suggest that CPRGS-1 is involved in regulation of Gα subunit CPG-1-mediated signaling and establish a role for a RGS protein in the modulation of virulence, conidiation, and hydrophobin synthesis in a plant pathogenic fungus

Establishing an in vivo assay system to identify components involved in environmental RNA interference in the western corn rootworm.

The discovery of environmental RNA interference (RNAi), in which gene expression is suppressed via feeding with double-stranded RNA (dsRNA) molecules, opened the door to the practical application of RNAi-based techniques in crop pest management. The western corn rootworm (WCR, Diabrotica virgifera virgifera) is one of the most devastating corn pests in North America. Interestingly, WCR displays a robust environmental RNAi response, raising the possibility of applying an RNAi-based pest management strategy to this pest. Understanding the molecular mechanisms involved in the WCR environmental RNAi process will allow for determining the rate limiting steps involved with dsRNA toxicity and potential dsRNA resistance mechanisms in WCR. In this study, we have established a two-step in vivo assay system, which allows us to evaluate the involvement of genes in environmental RNAi in WCR. We show that laccase 2 and ebony, critical cuticle pigmentation/tanning genes, can be used as marker genes in our assay system, with ebony being a more stable marker to monitor RNAi activity. In addition, we optimized the dsRNA dose and length for the assay, and confirmed that this assay system is sensitive to detect well-known RNAi components such as Dicer-2 and Argonaute-2. We also evaluated two WCR sid1- like (sil) genes with this assay system. This system will be useful to quickly survey candidate systemic RNAi genes in WCR, and also will be adaptable for a genome-wide RNAi screening to give us an unbiased view of the environmental/systemic RNAi pathway in WCR

Hypovirus papain-like protease p29 suppresses RNA silencing in the natural fungal host and in a heterologous plant system

Virulence-attenuating hypoviruses of the species Cryphonectria hypovirus 1 (CHV1) encode a papain-like protease, p29, that shares similarities with the potyvirus-encoded suppressor of RNA silencing HC-Pro. We now report that hypovirus CRV1-EP713-encoded p29 can suppress RNA silencing in the natural host, the chestnut blight fungus Cryphonectria parasitica. Hairpin RNA-triggered silencing was suppressed in C parasitica strains expressing p29, and transformation of a transgenic green fluorescent protein (GFP)-silenced strain with p29 resulted in an increased number of transformants with elevated GFP expression levels. The CHV1-EP713 p29 protein was also shown to suppress both virus-induced and agroinfiltration-induced RNA silencing and systemic spread of silencing in GFP-expressing transgenic Nicotiana benthamiana line 16c plants. The demonstration that a mycovirus encodes a suppressor of RNA silencing provides circumstantial evidence that RNA silencing in fungi may serve as an antiviral defense mechanism. The observation that a phylogenetically conserved protein of related plant and fungal viruses functions as a suppressor of RNA silencing in both fungi and plants indicates a level of conservation of the mechanisms underlying RNA silencing in these two groups of organisms

Physiological and cellular responses caused by RNAi- mediated suppression of Snf7 orthologue in western corn rootworm (Diabrotica virgifera virgifera) larvae.

Ingestion of double stranded RNA (dsRNA) has been previously demonstrated to be effective in triggering RNA interference (RNAi) in western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte), providing potential novel opportunities for insect pest control. The putative Snf7 homolog of WCR (DvSnf7) has previously been shown to be an effective RNAi target for insect control, as DvSnf7 RNAi leads to lethality of WCR larvae. Snf7 functions as a part of the ESCRT (Endosomal Sorting Complex Required for Transport) pathway which plays a crucial role in cellular housekeeping by internalization, transport, sorting and lysosomal degradation of transmembrane proteins. To understand the effects that lead to death of WCR larvae by DvSnf7 RNAi, we examined some of the distinct cellular processes associated with ESCRT functions such as de-ubiquitination of proteins and autophagy. Our data indicate that ubiquitinated proteins accumulate in DvSnf7 dsRNA-fed larval tissues and that the autophagy process seems to be impaired. These findings suggest that the malfunctioning of these cellular processes in both midgut and fat body tissues triggered by DvSnf7 RNAi were the main effects leading to the death of WCR. This study also illustrates that Snf7 is an essential gene in WCR and its functions are consistent with biological functions described for other eukaryotes

Evaluation of the assay system 1: dsRNA length-dependent competition.

<p>The length of the dsRNA used in the initial RNAi affects the efficiency of the subsequent second RNAi. Various lengths of KA dsRNA were used for the initial RNAi (1st RNAi), while 500 bp <i>lac2</i> dsRNA was used for the second “marker gene” RNAi (2nd RNAi). The initial number of the larvae, the number of the larvae that survived the assay, and the number of larvae that became the second instar are indicated in the table. The second instar larvae were analyzed for their phenotypes, and categorized into the Lac2 phenotype (<i>i.e.</i> the second RNAi worked) and the WT phenotype (<i>i.e.</i> the second RNAi was suppressed). The suppression of the second RNAi phenotype by the initial RNAi was evaluated as the proportion of the WT larvae in the survived second instar larvae (Suppression).</p

Progression of pathology in the midgut of <i>dsDvSnf7</i>-treated WCR larvae.

<p>Observed symptoms were summarized in four phases (1 = lightest to 4 = most severe). Highlighted are macroautophagic complexes (blue ellipses) (diagram, A′), gut lumen (L with orange glowing background) filled with cellular debris and loss of microvilli (white arrowheads) (diagrams, B, B″), large vacuoles (diagrams, B′, B″, C′) (green arrows), luminal membrane disintegration (black arrowheads) (diagram, C″) and infolding of the basal membrane (transparent black arrowheads) (diagrams, C″, D′, D″), progressive cell sloughing (asterisks) (diagrams, A″, B, C). Panels E-E″ show midgut of dsGFP-treated control larvae. Highlighted are endosomes (white arrows), lysosomes (magenta arrows) and autolysosomes (black arrow). refers to grey area denoting sectional artifact (cracks in the tissue section). Scale bars  = 4 µm (A-E) and 2 µm (A′-E′ and A″-E″).</p

Midgut ultrastructural profiles of untreated, dsGFP-treated and dsDvSnf7-treated western corn rootworm (WCR) second instar larvae fed for 5 days.

<p>The letter L with orange glowing background indicates the region of gut lumen, white arrows indicate endosomes, magenta arrows indicate lysosomes, black arrows indicate autolysosomes and MLB is multi-lamellar bodies. C′ is for the magnified view of macroautophagic complex in the midgut of dsDvSnf7-treated individuals. Scale bars  = 4 µm (a, b, c), 1 µm (a′, b′) and 500 nm (c′). D refers to macroautophagic complexes counted in enterocytes of untreated, dsGFP-treated and dsDvSnf7-treated larval midgut. Error bars represent mean ± S.D. of three individuals.</p

<i>lac2</i> and <i>ebony</i> feeding RNAi phenotypes in WCR.

<p>(A–C) wild-type, (D–F) KA dsRNA fed, (G–H) <i>lac2</i> RNAi, and (J–L) <i>ebony</i> RNAi. Both <i>lac2</i> and <i>ebony</i> RNAi affect the pigmentation seen in the larval head, legs, and the posterior-most segment.</p

Effect of dsRNA length on feeding RNAi efficiency.

<p>(A) wild-type, (B) KA dsRNA, (C–H) <i>lac2</i> RNAi with various lengths of dsRNA, (I–J) <i>ebony</i> RNAi with various lengths of dsRNA. Note that feeding dsRNA longer than 100 bp induced easily identifiable pigmentation defects both in <i>lac2</i> and <i>ebony</i> RNAi.</p