Expression profiles of cytokinin oxidase (CKX) genes in <i>Arabidopsis</i> plants after <i>Botrytis cinerea</i> inoculation.

Transcript levels of CKX family were determined by qRT-PCR in 4-weeks-old leaves inoculated with B. cinerea spores at different time-points, and taking leaves inoculated with the same amount of 1/2 PDB solution as the mock treatment. Expression levels in the mock leaves at 14 h post inoculation were set to a value of 1. All data were normalized to the expression of EXP (At4g26410). Asterisks indicate significant differences between B. cinerea and mock-treated samples at the same time-point (two-tailed Student’s t-test: *P < 0.05, **P < 0.01, ***P < 0.001). Error bars are standard deviations (n = 3). Three independent experiments were performed with a similar outcome; results from one representative experiment are shown.</p

Alteration of in <i>vivo EFR6</i> or <i>AHL15</i> levels changed the susceptibility of <i>Arabidopsis</i> plants and the response of <i>CKX5</i> to <i>Botrytis cinerea</i> infection.

(a-c) Leaves of 4-weeks-old wild type (WT) and erf6 mutant, ERF6 overexpression plants (ERF6OE), ahl15 mutant were inoculated with B. cinerea spores and the disease symptoms were analyzed two days later. Scale bars in photographs of the infected leaves are 1 cm. The values shown for disease lesion size in the indicated genotypes are means ± SD (n = 50 inoculated leaves). Asterisks indicate significant differences between WT and mutant or transgenic plants (two-tailed Student’s t-test, **P 4 mm plus chlorosis. The significance of differences was analyzed by χ2-test. **P CKX5 expression levels were analyzed in leaves of the four genotypes at 24 h and 48 h post-inoculation with B. cinerea or mock solution. Expression values in WT leaves with mock treatment were set to a value of 1. Bars are the means ± SD (n = 3). Three independent experiments were performed with similar outcomes; results from one representative experiment are shown. Asterisks indicate significant differences between WT and erf6, ERF6OE, or ahl15 with same treatments (two-tailed Student’s t-test, *P < 0.05; **P < 0.01).</p

Confirmation of the 13 screened transcription factors (TF) with pair-wise yeast one hybrid.

(a) Coding sequences (CDS) of the screened 13 TFs were separately cloned into the prey vector pGADT7 AD in frame with the activation domain (AD). The corresponding gene name and restriction sites used for cloning were shown. PADH1, constitutive ADH1 promoter; GAL4 AD, encodes GAL4 activation domain; LEU2, nutritional marker for selection in yeast. (b-d) The prey vectors were transformed into the corresponding bait strains and grown on SD medium lacking leucine (SD-Leu) with corresponding concentrations of AbA. The control stains contained empty pGADT7 AD and bait vector.</p

Transgenic <i>Arabidopsis</i> plants with overexpression of <i>CKX5</i> were more resistant to <i>Botrytis cinerea</i> infection.

(a) Relative expression levels of CKX5 in transgenic Arabidopsis. WT, wild type. CKX5OE, CKX5-overexpressing transgenic lines. (b) Leaves from 4-weeks-old WT and the three transgenic lines were photographed 2 days after B. cinerea inoculation. Scale bars = 1 cm. (c) Disease lesion size in the indicated genotypes. The values shown are the means ± SD (n = 50 inoculated leaves). Significant differences between WT and transgenic lines were analyzed by two-tailed Student’s t-test. **P d) Disease symptoms of leaves from the indicated genotypes. Class I, lesion 4 mm plus chlorosis. The distribution was calculated from 50 leaves. The significance of differences was analyzed by χ2-test. **P < 0.01; ***P < 0.001.</p

Generation of bait strains for yeast one-hybrid (Y1H) screening.

(a) Physical map of the bait plasmids used for integration into yeast genome. Five short overlapping promoter fragments of CKX5 were separately inserted into the vector of pAbAi through HindIII and XhoI restriction sites. AurR, encodes aureobasidin A-resistant mutant of inositol phosphorylceramide synthase, confers fungal resistance to Aureobasidin A (AbA); AmpR, confers resistance to ampicillin, carbenicillin, and related antibiotics; URA3, encodes orotidine5’-phosphatedecarboxylase. (b) Determination the minimal inhibitory concentrations of AbA for the bait strains. The numbers on the right side of AbA represent the concentration of AbA with unit of ng/mL. SD-Ura means synthetically defined medium lacking uracil.</p

Mutation of the binding sequences repressed the interactions between WRKY40, WRKY33, SPL3 and <i>CKX5</i> promoter fragments.

(a) Physical map of the CKX5 promoter illustrating the changes of binding sites for WRKY40, WRKY33 and SPL3. The arrows showed how the original sequences were changed. (b) Determination of the interactions between the three transcription regulators and the mutated promoter of CKX5 with yeast one-hybrid assays. Y1H Gold strains successfully transformed with corresponding vectors were grown on the SD-Leu/AbA plates at 30 °C for 3–5 days. PCKX5-2 or PCKX5-5 represents the original fragment of CKX5 promoter. PMuCKX5-2 or PMuCKX5-5 means the fragments with mutated binding sites. The numbers on the right side of AbA represent the concentration of AbA with unit of ng/mL.</p

Expression levels of selected transcription regulator genes in response to <i>Botrytis cinerea</i>.

Transcript accumulations of the genes were determined by real-time qPCR in 4-weeks-old wild type Arabidopsis leaves inoculated with B. cinerea at different time-points. Leaves inoculated with same amount of 1/2 PDB solution as mock. Expression levels in the mock leaves at 14 h post-inoculation (hpi) were set to a value of 1. Error bars are standard deviations (n = 3). Differences are significant at P t-test.</p

Primers used for quantitative real-time PCR.

In our previous work, cytokinin (CK) signaling and biosynthesis were found to be modulated during Arabidopsis defense against infection by the necrotrophic pathogen Botrytis cinerea. Notably, the expression level of CYTOKININ OXIDASE/DEHYDROGENASE 5 (CKX5) was significantly induced in B. cinerea-infected leaves and later in distant B. cinerea-untreated leaves of the same plant. To confirm and determine how CKX5 is involved in the response to B. cinerea infection, transcript levels of CKX family genes were analyzed in B. cinerea-inoculated leaves, and only CKX5 was remarkably induced by B. cinerea infection. Furthermore, CKX5-overexpressing Arabidopsis plants were more resistant to B. cinerea than wild-type plants. Transcription factors (TFs) binding to the CKX5 promoter were then screened by yeast one-hybrid assays. Quantitative Real-Time Reverse Transcription PCR (qRT-PCR) analysis further showed that genes encoding TFs, including WRKY40, WRKY33, ERF6, AHL15, AHL17, ANAC003, TCP13 and ANAC019, were also strongly induced in infected leaves, similar to CKX5. Analysis of ERF6-overexpressing plants and ERF6-and AHL15-knockout mutants indicated that ERF6 and AHL15 are involved in plant immunity to B. cinerea. Furthermore, CKX5 upregulation by B. cinerea infection was affected when ERF6 or AHL15 levels were altered. Our work suggests that CKX5 levels are controlled by the plant defense system to defend against attack by the pathogen B. cinerea.</div

Phenotypes of wild type (<i>WT</i>) and transgenic <i>Arabidopsis</i> plants overexpressing <i>CKX5</i> (<i>CKX5OE</i>).

(a) Seed germination assays on medium containing 30 mg/L hygromycin confirm the homozygosity of the transgenic lines. (b) 4 weeks-old wild type and transgenic plants CKX5OE grown under photoautotrophic conditions in soil. Bars in (a) and (b) are 1 cm. (JPG)</p

The information for selected transcription factors screened by yeast one-hybrid.

The information for selected transcription factors screened by yeast one-hybrid.</p