Microbial symbionts in insects influence down-regulation of defense genes in maize

Abstract

Diabrotica virgifera virgifera larvae are root-feeding insects and significant pests to maize in North America and Europe. Little is known regarding how plants respond to insect attack of roots, thus complicating the selection for plant defense targets. Diabrotica virgifera virgifera is the most successful species in its genus and is the only Diabrotica beetle harboring an almost species-wide Wolbachia infection. Diabrotica virgifera virgifera are infected with Wolbachia and the typical gut flora found in soil-living, phytophagous insects. Diabrotica virgifera virgifera larvae cannot be reared aseptically and thus, it is not possible to observe the response of maize to effects of insect gut flora or other transient microbes. Because Wolbachia are heritable, it is possible to investigate whether Wolbachia infection affects the regulation of maize defenses. To answer if the success of Diabrotica virgifera virgifera is the result of microbial infection, Diabrotica virgifera virgifera were treated with antibiotics to eliminate Wolbachia and a microarray experiment was performed. Direct comparisons made between the response of maize root tissue to the feeding of antibiotic treated and untreated Diabrotica virgifera virgifera show down-regulation of plant defenses in the untreated insects compared to the antibiotic treated and control treatments. Results were confirmed via QRT-PCR. Biological and behavioral assays indicate that microbes have integrated into Diabrotica virgifera virgifera physiology without inducing negative effects and that antibiotic treatment did not affect the behavior or biology of the insect. The expression data and suggest that the pressure of microbes, which are most likely Wolbachia, mediate the down-regulation of many maize defenses via their insect hosts. This is the first report of a potential link between a microbial symbiont of an insect and a silencing effect in the insect host plant. This is also the first expression profile for a plant attacked by a root-feeding insect.

Figure 1. Classification of 500 genes with the most significant differential expression.

500 genes with the most significant differential expression were categorized using a combination of gene annotation, gene ontology and scientific publication. 225 genes were related to plant defense and stress response, 126 genes were associated with metabolic processes, 30 were categorized as being involved with plant architecture and/or development, 44 genes were associated with DNA replication and 73 genes could not be classified as their functions are as of yet, unknown.

Figure 2. Expression pattern of untreated and antibiotic treated WCR.

The relative expression of the untreated and antibiotic treated WCR treatments was evaluated in respect to the control treatment. The untreated WCR treatment exhibited a down-regulation for 369 of the 500 genes. The antibiotic treated WCR showed and up-regulation of 343 of the 500 genes.

Figure 3. The relationship of the 500 most significant genes between the 3 treatments.

A Ven diagram representation of the distribution of the 500 most significant genes indicates that no genes displayed similar expression for all 3 treatments. The control treatment shared similar expression of 89 genes with the treated WCR treatment and 181 genes with the untreated WCR treatment. The WCR treatments did not share similar expression for any of the 500 genes.

Figure 4. Expression profile of maize defense genes from TIGR Multiple Array Viewer.

The expression profile of the data represented as a heat map illustrates that feeding by untreated WCR resulted in down-regulation of plant defense to levels below that of the non-feeding control and up-regulated following attack of antibiotic treated WCR. The column designated as WCR + Wolbachia represents WCR without antibiotic treatment. Likewise, the column designated as WCR represents WCR that were treated with tetracycline. (A) PR proteins, (B) Phytoalexins, (C) Cell wall associated factors. Green indicates gene down-regulation while red indicates gene up-regulation.

Figure 5. The effects of microbes on WCR fitness.

A hatch assay was conducted in from an isolated sample of eggs; both colonies display a bell shaped hatch distribution in relation to time. T-tests for number hatched and rate of hatch are statistically not significant. Diamonds represent WCR that were treated with tetracycline while squares represent WCR without antibiotic treatment.

Figure 6. The effects of microbes on WCR larval competitiveness.

A host location assay was conducted to determine the effect of microbial infection on WCR larval competitiveness. T-tests for number of WCR that were able to locate the host plant and rate of host location are statistically not significant. Diamonds represent WCR that were treated with tetracycline while squares represent WCR without antibiotic treatment.