Induction of PR-10 genes and metabolites in strawberry plants in response to Verticillium dahliae infection

Abstract

Background: The soil-borne vascular pathogen Verticillium dahliae causes severe wilt symptoms in a wide range of plants including strawberry (Fragaria × ananassa). To enhance our understanding of the effects of V. dahliae on the growth and development of F. × ananassa, the expression patterns of 21 PR-10 genes were investigated by qPCR analysis and metabolite changes were determined by LC-MS in in vitro F. × ananassa plants upon pathogen infection.

Results: The expression patterns of the 21 isoforms showed a wide range of responses. Four PR-10 genes were highly induced in leaves upon pathogen infection while eight members were significantly up-regulated in roots. A simultaneously induced expression in leaves and roots was detected for five PR-10 genes. Interestingly, two isoforms were expressed upon infection in all three tissues (leaves, roots and stems) while no induction was detected for two other members. Accumulation of antifungal catechin and epicatechin was detected upon pathogen infection in roots and stems at late stages, while caffeic acid and citric acid were observed only in infected roots. Production of abscisic acid, salicylic acid, jasmonic acid (JA), gibberellic acid and indole acetic acid (IAA) was induced in infected leaves and stems at early stages. IAA and JA were the sole hormones to be ascertained in infected roots at late stages.

Conclusions: The induction of several PR-10 genes upon infection of strawberry plants with V. dahliae suggest a role of PR-10 genes in the defense response against this pathogen. Production of phytohormones in the early stages of infection and antifungal metabolites in late stages suppose that they are implicated in this response. The results may possibly improve the control measures of the pathogen.

Keywords: Fragaria × ananassa; Gene expression; PR-10; Strawberry; Verticillium dahliae; qPCR.

Fig. 1

Development of disease symptoms in in vitro plants infected with V. dahliae at different time points (1, 5, 10, 20, and 30 days) and comparison of methanol extracts of lyophilized tissues from control and infected organs at different time points post V. dahliae infection (1, 5, 10, 20 and 30 days)

Fig. 2

Relative transcript levels determined by qPCR of PR-10.03, PR-10.11, PR-10.16 and PR-10.21, which are highly induced in leaves. Strawberry plants were infected with V. dahliae and the relative transcript levels were normalized to the control stem at 1 dpi (set to one). The expression levels in the tissues were measured at 1, 5, 10, 20, and 30 dpi (days post infection). Green bars represent the control while red bars represent infected tissues. The mean values (± SD) were obtained from five biological replicates and are shown as relative changes. Asterisks represent significant induction of a gene after statistical comparison with the transcript abundance of the control sample using the Tukey test. FaRIB413 was used as a housekeeping gene for normalization

Fig. 3

Relative transcript levels determined by qPCR of PR-10.06, PR-10.08, PR-10.17, PR-10.02, PR-10.20, PR-10.14, PR-10.15 and PR-10.18, which are highly induced in roots. Strawberry plants were infected with V. dahliae and the relative transcript level was normalized to the control stem at 1 dpi (set to one). The expression levels in the tissues were measured at 1, 5, 10, 20, and 30 dpi (days post infection). Green bars represent the control while red bars represent infected tissues. The mean values (± SD) were obtained from five biological replicates and are shown as relative changes. Asterisks represent significant induction of a gene after statistical comparison with the transcript abundance of the control sample using the Tukey test. FaRIB413 was used as a housekeeping gene for normalization

Fig. 4

Relative transcript levels determined by qPCR of PR-10.04, PR-10.01, PR-10.12, PR-10.19 and PR-10.10 which are highly induced in leaves and roots. Strawberry plants were infected with V. dahliae and the relative transcript level was normalized to the control stem at 1 dpi (set to one). The expression levels in the tissues were measured at 1, 5, 10, 20, and 30 dpi (days post infection). Green bars represent the control while red bars represent infected tissues. The mean values (± SD) were obtained from five biological replicates and are shown as relative changes. Asterisks represent significant induction of a gene after statistical comparison with the transcript abundance of the control sample using the Tukey test. FaRIB413 was used as a housekeeping gene for normalization

Fig. 5

Relative transcript levels determined by qPCR of PR-10.13 and PR-10.09, which are highly induced in leaves, roots, and stems and relative transcript levels of PR-10.05 and PR-10.07 which indicated no induction for both isoforms. Strawberry plants were infected with V. dahliae and the relative transcript level was normalized to the control stem at 1 dpi (set to one). The expression levels in the tissues were measured at 1, 5, 10, 20, and 30 dpi (days post infection). Green bars represent the control while red bars represent infected tissues. The mean values (± SD) were obtained from five biological replicates and are shown as relative changes. Asterisks represent significant induction of a gene after statistical comparison with the transcript abundance of the control sample using the Tukey test. FaRIB413 was used as a housekeeping gene for normalization

Fig. 6

Relative concentration (± SD) of metabolites and pytohormones in infected (with V. dahliae) and uninfected Fragaria tissues at 1, 5, 10, 20, and 30 dpi (days post infection). Relative concentrations (Rel. conc.) are expressed in mg-equ./g dry weight of the internal standard biochanin A in freeze-dried strawberry tissues (leaves, stems and roots). Asterisks (*) indicate significant differences for each metabolite after statistical comparison with its abundance in the control sample using the Tukey test