Use of BABA and INA As Activators of a Primed State in the Common Bean (Phaseolus vulgaris L.)

Abstract

To survive in adverse conditions, plants have evolved complex mechanisms that "prime" their defense system to respond and adapt to stresses. Their competence to respond to such stresses fundamentally depends on its capacity to modulate the transcriptome rapidly and specifically. Thus, chromatin dynamics is a mechanism linked to transcriptional regulation and enhanced defense in plants. For example, in Arabidopsis, priming of the SA-dependent defense pathway is linked to histone lysine methylation. Such modifications could create a memory of the primary infection that is associated with an amplified gene response upon exposure to a second stress-stimulus. In addition, the priming status of a plant for induced resistance can be inherited to its offspring. However, analyses on the molecular mechanisms of generational and transgenerational priming in the common bean (Phaseolus vulagris L.), an economically important crop, are absent. Here, we provide evidence that resistance to P. syringae pv. phaseolicola infection was induced in the common bean with the synthetic priming activators BABA and INA. Resistance was assessed by evaluating symptom appearance, pathogen accumulation, changes in gene expression of defense genes, as well as changes in the H3K4me3 and H3K36me3 marks at the promoter-exon regions of defense-associated genes. We conclude that defense priming in the common bean occurred in response to BABA and INA and that these synthetic activators primed distinct genes for enhanced disease resistance. We hope that an understanding of the molecular changes leading to defense priming and pathogen resistance will provide valuable knowledge for producing disease-resistant crop varieties by exposing parental plants to priming activators, as well as to the development of novel plant protection chemicals that stimulate the plant's inherent disease resistance mechanisms.

Keywords: BABA; INA; common bean; epigenetics; priming.

Figure 1

Lesion development in leaves from P. vulgaris plants inoculated with P. syringae pv. phaseolicola NPS 3121 (PspNPS3121) after treatment with (A) INA, (B) BABA, or (C) water; (D) Non-inoculated, untreated control plants. Photos were taken 10 days after pathogen inoculation.

Figure 2

(A) Lesion size and (B) colony-forming units (CFU) of P. vulgaris plants 10 days after inoculation with P. syringae (P.s). Control (ctrl) plants were neither treated with activator nor inoculated. Data represent mean ± SD, n = 3 independent experiments; data were analyzed with an unpaired two-tailed Student's t-test (^**p < 0.01, ^***p < 0.001).

Figure 3s

Transcript levels of genes from P. vulgaris involved in plant defense as determined by qRT-PCR at various days after germination (dag). Plants were primed with activators (BABA- or INA-treated plants) followed by inoculation with P. syringae pv. phaseolicola (Activator + P.s.), inoculated only (no activator + P.s.), or neither primed nor inoculated (control, ctrl). Data were normalized to the Actin11 (PvActin11) reference gene. (A) PvPR1; (B) PvPR4; (C) PvNPR1; (D) PvWRKY29; (E) PvWRKY53; (F) PvWRKY6. Data represent mean ± SD, n = 3 independent experiments. Statistical significance for the F0 generation was determined with multiple Student's t-test, followed by the Holm-Šídák multiple comparison test at a significance value of 0.05 (^*p < 0.05), by using the GraphPad Prism (v 6.0, GraphPad Software, San Diego California USA, www.graphpad.com) (see Supplementary Table 1).

Figure 4

Schematic representation of the P. vulgaris genes analyzed by ChIP. Black boxes represent exons, horizontal lines represent introns, red bars at the promoter-exon boundary region show the segments amplified by PCR, and the bent arrows represent the transcription start site.

Figure 5

Histone methylation profiles of the P. vulgaris genes involved in plant defense at various days after germination (dag). Plants were primed with activators and later inoculated with P. syringae pv. phaseolicola (Activator + P.s.), inoculated only (no activator + P.s.), or neither primed nor inoculated (control, ctrl). ChIP assays with antibodies specific for H3K4me3 in BABA-primed and INA-primed plants. (A) PvPR1; (B) PvPR4; (C) PvNPR1; (D) PvWRKY29; (E) PvWRKY53; (F) PvWRKY6. Depletion of H3K4me3 from the promoter-exon boundary region correlates with enhanced transcription of the primed genes. Two independent biological assays are shown.

Figure 6

Disease symptoms in unprimed, 2-week-old F1 progeny of P. vulgaris plants inoculated with P. syringae pv. phaseolicola (P.s.). F0 were: primed with activators and inoculated (Activator + P.s.), unprimed and inoculated (− + P.s), or neither primed nor inoculated. (A–D) photos of F1 leaves after inoculation; (E) Lesion size; and (F) CFUs. Data represent mean ± SD, n = 3 independent experiments; data were analyzed with an unpaired two-tailed Student's t-test (^*p < 0.05, ^**p < 0.01).

Figure 7

Transcript levels in F1 progeny of selected genes involved in plant defense. Progeny were either: unprimed and not inoculated (−) or unprimed and inoculated with P. syringae pv. phaseolicola (− + P.s.). F1 progeny were descended from F0 plants that had been primed with activator and inoculated with P. syringae pv. phaseolicola (Activator + P.s.), inoculated only (− + P.s.), or neither primed nor inoculated (Ctrl 1 or 2). Data were normalized to the Actin11 (PvActin11) reference gene. (A) PvWRKY6; (B) PvWRKY29; (C) PvWRKY53; (D) PvPR1; (E) PvPR4; (F) PvNPR1. Data represent mean ± SD, n = 3 independent experiments. Statistical significance for the F1 generation was determined with multiple Student's t-test, followed by the Holm-Šídák multiple comparison test at a significance value of 0.05 (^*p < 0.05), by using the GraphPad Prism (v 6.0, GraphPad Software, San Diego California USA, www.graphpad.com). A one-way ANOVA with Dunnett's post-test was performed using GraphPad Prism (v 6.0, GraphPad Software, San Diego California, USA) at a significance value of 0.05 (see Supplementary Table 2).

Figure 8

Histone methylation profiles as determined by ChIP assays with antibodies specific for H3K4me3. F1 progeny were descended from F0 plants that had been primed with activator and inoculated with P. syringae pv. phaseolicola (Activator + P.s.), inoculated only (− + P.s.), or neither primed nor inoculated (Ctrl). (A) PvPR1; (B) PvPR4; (C) PvNPR1; (D) PvWRKY29; (E) PvWRKY53; (F) PvWRKY6. Two independent biological assays are shown.