Overexpression of HvIcy6 in Barley Enhances Resistance against Tetranychus urticae and Entails Partial Transcriptomic Reprogramming

Abstract

Cystatins have been largely used for pest control against phytophagous species. However, cystatins have not been commonly overexpressed in its cognate plant species to test their pesticide capacity. Since the inhibitory role of barley HvCPI-6 cystatin against the phytophagous mite Tetranychus urticae has been previously demonstrated, the purpose of our study was to determine if barley transgenic lines overexpressing its own HvIcy6 gene were more resistant against this phytophagous infestation. Besides, a transcriptomic analysis was done to find differential expressed genes among wild-type and transformed barley plants. Barley plants overexpressing HvIcy6 cystatin gene remained less susceptible to T. urticae attack when compared to wild-type plants, with a significant lesser foliar damaged area and a lower presence of the mite. Transcriptomic analysis revealed a certain reprogramming of cellular metabolism and a lower expression of several genes related to photosynthetic activity. Therefore, although caution should be taken to discard potential deleterious pleiotropic effects, cystatins may be used as transgenes with impact on agricultural crops by conferring enhanced levels of resistance to phytophagous pests.

Keywords: Tetranychus urticae; barley; cystatin; plant defense; transcriptomics; transgenic.

Figure 1

Messenger expression levels of HvIcy6 gene in barley wild-type plants after T. urticae infestation, assayed by RT-qPCR. Total RNA was extracted at seven (7) or fourteen (14) days after treatment from leaves of infested or non-infested plants. Data were expressed as mRNA levels normalized to barley cyclophilin mRNA content. Different letters (a,b) indicate significant differences (p < 0.05, One-Way ANOVA Student Newman-Keuls test).

Figure 2

Leaf damage in wild-type and OE Icy6 lines (1019 and 1082) after 7 days of T. urticae infestation. (a) Phenotypic aspect of the plants. (b) Quantification of leaf feeding damage. Damage was measured as mm² of injured foliar area of all leaves. (c) Total number of leaves per plant. Data from six independent plants. Different letters (a,b) indicate significant differences (p < 0.05, One-Way ANOVA Student Newman-Keuls SNK test). (d) Examples of leaves from transgenic and non-transgenic plants, numbered from L1 (oldest leaf) to L4 (youngest leaf) at this stage of development. Regions of L1 and L2 leaves are magnified to highlight chlorotic spots due to mite feeding.

Figure 3

Leaf damage in wild-type and OE Icy6 lines (1019 and 1082) after 14 days of T. urticae infestation. (a) Phenotypic aspect of the plants. (b) Quantification of leaf yellowish measured as cm² of yellow-brown foliar area of all leaves. (c) Total number of leaves per plant. Data from six independent plants. Different letters (a,b) indicate significant differences (p < 0.05, One-Way ANOVA Student Newman-Keuls SNK test). (d) Examples of all the leaves from individual transgenic and non-transgenic plants.

Figure 4

Messenger expression levels of TuRp49 gene in barley wild-type and OE Icy6 plants after T. urticae infestation, assayed by RT-qPCR. Total RNA was extracted at seven or fourteen days after infestation. Data were expressed as mRNA levels normalized to barley cyclophilin mRNA content. Different letters (a,b,c) indicate significant differences (p < 0.05, One-Way ANOVA Student Newman-Keuls test).

Figure 5

Analysis of the differentially expressed genes (DEGs) among WT and OE Icy6 plants. (a) Heatmap showing normalized colour intensity from the expression values of the DEGs in the three independent replicates. Green represents up-regulation and red represents down-regulation. (b) Circle charts showing the percentage on up- or down-regulated DEGs for the main representative categories in each subset.

Figure 5

Analysis of the differentially expressed genes (DEGs) among WT and OE Icy6 plants. (a) Heatmap showing normalized colour intensity from the expression values of the DEGs in the three independent replicates. Green represents up-regulation and red represents down-regulation. (b) Circle charts showing the percentage on up- or down-regulated DEGs for the main representative categories in each subset.