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Citrus leprosis virus C Infection Results in Hypersensitive-Like Response, Suppression of the JA/ET Plant Defense Pathway and Promotion of the Colonization of Its Mite Vector - PubMed

  • ️Fri Jan 01 2016

Citrus leprosis virus C Infection Results in Hypersensitive-Like Response, Suppression of the JA/ET Plant Defense Pathway and Promotion of the Colonization of Its Mite Vector

Gabriella D Arena et al. Front Plant Sci. 2016.

Abstract

Leprosis is a serious disease of citrus caused by Citrus leprosis virus C (CiLV-C, genus Cilevirus) whose transmission is mediated by false spider mites of the genus Brevipalpus. CiLV-C infection does not systemically spread in any of its known host plants, thus remaining restricted to local lesions around the feeding sites of viruliferous mites. To get insight into this unusual pathosystem, we evaluated the expression profiles of genes involved in defense mechanisms of Arabidopsis thaliana and Citrus sinensis upon infestation with non-viruliferous and viruliferous mites by using reverse-transcription qPCR. These results were analyzed together with the production of reactive oxygen species (ROS) and the appearance of dead cells as assessed by histochemical assays. After interaction with non-viruliferous mites, plants locally accumulated ROS and triggered the salicylic acid (SA) and jasmonate/ethylene (JA/ET) pathways. ERF branch of the JA/ET pathways was highly activated. In contrast, JA pathway genes were markedly suppressed upon the CiLV-C infection mediated by viruliferous mites. Viral infection also intensified the ROS burst and cell death, and enhanced the expression of genes involved in the RNA silencing mechanism and SA pathway. After 13 days of infestation of two sets of Arabidopsis plants with non-viruliferous and viruliferous mites, the number of mites in the CiLV-C infected Arabidopsis plants was significantly higher than in those infested with the non-viruliferous ones. Oviposition of the viruliferous mites occurred preferentially in the CiLV-C infected leaves. Based on these results, we postulated the first model of plant/Brevipalpus mite/cilevirus interaction in which cells surrounding the feeding sites of viruliferous mites typify the outcome of a hypersensitive-like response, whereas viral infection induces changes in the behavior of its vector.

Keywords: Arabidopsis; Brevipalpus mites; Citrus sinensis; RNA silencing; cilevirus; herbivory; hormonal crosstalk; plant-virus-vector interaction.

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Figures

Figure 1
Figure 1

Phenotypes of Arabidopsis thaliana (left) and Citrus sinensis (right) plants infested with non-viruliferous and CiLV-C viruliferous Brevipalpus yothersi mites during the time course experiments. Pictures from Arabidopsis and C. sinensis were taken at 8 and 25 days after infestation, respectively. Infested leaves are indicated by asterisks (*).

Figure 2
Figure 2

Expression profiles of Arabidopsis thaliana genes involved in the main plant defense pathways assessed by RT-qPCR. In green, plants kept without mites. In orange, plants infested with non-viruliferous mites. In red, plants infested with CiLV-C viruliferous mites. Data are presented as fold change values in comparison with not infested plants (with fold change value set to 1) or expression levels when transcripts were not detected in not infested plants. Values represent the mean of 10 biological replicates for each set. Error bars represent standard errors. Different letters correspond to different expression levels between treatments within the same time point (ANOVA and Tukey's HSD post-hoc test, α < 0.05). hai, hours after infestation; dai, days after infestation.

Figure 3
Figure 3

CiLV-C loads on Arabidopsis thaliana (A) and Citrus sinensis (B) plants infested with viruliferous Brevipalpus yothersi mites throughout the time course of the interaction. Data are presented as absolute number of copies (log) of the CiLV-C p29 gene normalized against SAND genes corresponding to each plant species. Values represent the means of 10 biological replicates for each time point. Standard errors are represented on each bars. Different letters correspond to different number of copies between the time points assessed (ANOVA and Tukey's HSD post-hoc test, α < 0.05).

Figure 4
Figure 4

Expression profiles of Citrus sinensis genes involved in the main plant defense pathways assessed by RT-qPCR. In green, plants kept without mites. In orange, plants infested with non-viruliferous mites. In red, plants infested with CiLV-C viruliferous mites. Data are presented as fold change values in comparison with not infested plants (with fold change value set to 1). Values represent the mean of 20 biological replicates for each set. Error bars represent standard errors. Different letters correspond to different expression levels between treatments within the same time point (ANOVA and Tukey's HSD post-hoc test, α < 0.05). hai, hours after infestation; dai, days after infestation.

Figure 5
Figure 5

Accumulation of reactive oxygen species (ROS) and cell death in Arabidopsis thaliana plants infested with non-viruliferous and CiLV-C viruliferous Brevipalpus yothersi mites at 6 hours after the infestation (hai), 12 hai, 24 hai, and 8 days after the infestation (dai). (A) Detection of hydrogen peroxide by 3,3′-diaminobenzidine (DAB) staining at 8 dai on leaves infested with viruliferous mites, non-viruliferous mites and not infested, respectively. Highlights on leaves midribs represent the regions where mites concentrate, used to quantification of stained area. (B) Quantitative measure of DAB brownish areas on highlighted regions along the course of the infestation. Values represent the means of 10 biological replicates for each set. Error bars represent standard errors. (C) Detail of ROS detection in leaves stained with DAB. (D) Detail of cell death in leaves stained with trypan blue.

Figure 6
Figure 6

Distribution of Brevipalpus yothersi adult mites and eggs in Arabidopsis thaliana. Non viruliferous (NV) or CiLV-C viruliferous (V) mites were deposited in three leaves of each plant and were counted at 13 days after the infestation. Data are presented as the average percentages of mites or eggs in the primarily infested leaves (where mites were initially deposited) and secondary infested leaves (due to mite migration to contiguous leaves). Error bars represent standard errors. Statistically significant differences at p < 0.05 (*) or < 0.01 (**) are indicated. Bracket indicates the comparison between the number of NV and V mites.

Figure 7
Figure 7

Model representing the interaction of Arabidopsis thaliana plants/Brevipalpus mites/CiLV-C. Left and right leaf halves show hallmarks during the plant interaction with non-viruliferous and viruliferous mites, respectively. During the feeding of non-viruliferous mites, saliva and putative mite-encoded effectors are delivered into the mesophyll cells targeted by the mite's stylet. Reactive oxygen species (ROS) burst, localized cell death and SA and JA/ET pathways are triggered. Mite induces the expression of the ERF-branch responsive gene PDF1.2 rather than the MYC-branch responsive gene VSP2, which might restrain plant response against herbivores. In plant infested by CiLV-C viruliferous mites, virions reach mesophyll cells mixed with the salivary flow. CiLV-C multiplies in the initially infected cell and locally moves to the neighboring cells. Mite feeding and the increasing viral loads generate a stronger stimulus which intensifies the ROS production, the number of dead cells around the mite feeding site and the SA-mediated response. Increased activity of the SA pathway likely promotes the inhibition of the JA/ET pathways, leading the downregulation of PDF1.2 and VSP2, which probably improve the mite performance. Both non-viruliferous and viruliferous mite infestations induce expression profile changes in the core genes of the gene silencing mechanism. Enhanced SA signaling activity in viral presence may contribute to higher RDR1 expression. A putative virus suppressor of RNA silencing (VSR) may target and inactivate AGO1, leading to the up-regulation of AGO2, which addresses a second antiviral defense line of the RNA silencing. CiLV-C remains restricted at cells surrounding the inoculation site and chlorosis symptoms develop seven to 10 days after inoculation, probably as a result of a hypersensitive-like response (HR) as consequence of an incompatible interaction.

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