Expression of the maize ZmGF14-6 gene in rice confers tolerance to drought stress while enhancing susceptibility to pathogen infection

Abstract

14-3-3 proteins are found in all eukaryotes where they act as regulators of diverse signalling pathways associated with a wide range of biological processes. In this study the functional characterization of the ZmGF14-6 gene encoding a maize 14-3-3 protein is reported. Gene expression analyses indicated that ZmGF14-6 is up-regulated by fungal infection and salt treatment in maize plants, whereas its expression is down-regulated by drought stress. It is reported that rice plants constitutively expressing ZmGF14-6 displayed enhanced tolerance to drought stress which was accompanied by a stronger induction of drought-associated rice genes. However, rice plants expressing ZmGF14-6 either in a constitutive or under a pathogen-inducible regime showed a higher susceptibility to infection by the fungal pathogens Fusarium verticillioides and Magnaporthe oryzae. Under infection conditions, a lower intensity in the expression of defence-related genes occurred in ZmGF14-6 rice plants. These findings support that ZmGF14-6 positively regulates drought tolerance in transgenic rice while negatively modulating the plant defence response to pathogen infection. Transient expression assays of fluorescently labelled ZmGF14-6 protein in onion epidermal cells revealed a widespread distribution of ZmGF14-6 in the cytoplasm and nucleus. Additionally, colocalization experiments of fluorescently labelled ZmGF14-6 with organelle markers, in combination with cell labelling with the endocytic tracer FM4-64, revealed a subcellular localization of ZmGF14-6 in the early endosomes. Taken together, these results improve our understanding of the role of ZmGF14-6 in stress signalling pathways, while indicating that ZmGF14-6 inversely regulates the plant response to biotic and abiotic stresses.

Fig. 1.

Expression analysis of ZmGF14-6 in maize. qRT-PCR was used to monitor ZmGF14-6 transcript abundance in maize tissues. The cyclophilin gene (X68678) was used as the internal control for normalization. Data represent mean ± SD (n = 16) from one biological experiment. These experiments were repeated three times with similar results. (A) Expression pattern of ZmGF14-6 in response to fungal infection. Germinating maize embryos were inoculated with spores from the fungus F. verticillioides and harvested at the indicated times after inoculation. (B) ZmGF14-6 gene expression in response to salt treatment. Roots of 7-day-old seedlings were treated with 200 mM NaCl and harvested at the indicated times. (C) Expression pattern of ZmGF14-6 in response to PEG-induced drought stress. Roots of 7-day-old seedlings were treated with 20% PEG 8000, a stress treatment commonly used to mimic drought conditions. ZmGF14-6 transcript levels were measured at the indicated times of treatment.

Fig. 2.

Molecular and phenotypic characterization of rice plants expressing ZmGF14-6. (A) Northern blot analysis of transgenic rice plants (T2 generation) expressing the ZmGF14-6 gene under the control of the maize constitutive promoter ubiquitin (Ubi::ZmGF14-6) or the pathogen- and wound-inducible promoter of the maize PR4 gene (ZmPR4::ZmGF14-6). In each case, three independently generated transgenic lines are presented (Ubi::ZmGF14-6, lines 4, 9, and 14; ZmPR4::ZmGF14-6, lines 1, 8, and 10). As for ZmPR4::ZmGF14-6 lines, leaves were mechanically wounded prior Northern blot analysis (denoted by asterisks). Samples from wounded leaves were harvested 24 h after wounding. Total RNAs (10 μg) were subjected to formaldehyde-containing agarose gel electrophoresis and hybridized with a ³²P-labelled probe. Lane WT, untransformed Senia control. Lane pC, rice plants transformed with the empty pCAMBIA1300 vector. Lower panels show ethidium bromide staining of RNA samples. (B) Immunoblot analysis of total protein extracts from transgenic rice plants constitutively expressing the ZmGF14-6 gene. Leaf protein extracts (20 μg) were separated by 12.5% SDS/PAGE, transferred to nitrocellulose membranes, and probed with the anti-ZmGF14-6 antibody. The RuBisCO protein served as a loading control. (C) Mean height of ZmGF14-6 rice plants (T2 homozygous lines) compared with wild-type (WT) plants. WT and transgenic lines, Ubi::ZmGF14-6, and ZmPR4::ZmGF14-6, as well as vector-transformed (pC) plants, were grown in the greenhouse for 3 weeks. Means were calculated from three independent transgenic lines. The data represent means ± SD (n = 16). (D–F) Productivity of rice plants constitutively expressing ZmGF14-6 (Ubi:ZmGF14-6). Seed production (D), number of spikes (E), and weight of seeds (F) in Ubi:ZmGF14-6, WT, and empty vector (pC) rice plants. For determination of seed weight, seeds from three independent lines were collected from three plants and pooled. Two hundred seeds from each pool were weighted and the mean weight per seed was calculated. The data represent mean ± SD.

Fig. 3.

Phenotype of ZmGF14-6 rice plants under drought stress conditions. (A) Diagram showing conditions used for drought tolerance assay. Plants were grown under well-watered conditions for 15 days. For drought stress treatment, water was withheld for 18 days (starting at D0) and then plants were supplied with water again for 7 days. In each experiment, wild-type (WT), empty-vector (pC), and transgenic lines (lines 4, 9 and 14), and at least 36 plants per line, were assayed. Assays to determine the tolerance to drought stress were carried out three times and at different developmental stages with similar results. (B) Phenotype of non-transformed (WT), lines transformed with the empty vector (pC), and lines constitutively expressing the ZmGF4-6 gene (lines 4, 9 and 14) grown under normal irrigation conditions for 15 days (panel D0). The pictures were taken at 14 and 18 days of drought stress (D14 and D18, respectively), and after 7 days after rewatering (RW). (C) Survival of ZmGF14-6 rice plants after 7 days of recovery from drought stress. Transgenic plants show 100% survival. Each column represents the mean ± SD of triplicate experiments (n = 36 in each experiment) (D) Water loss in wild-type and transgenic rice plants. Leaves were harvested from wild-type and ubi::ZmGF14-6 rice plants (n = 15) at the three-leaf stage. Fresh weight of detached leaves was measured (time 0). Water loss was calculated from the decrease in fresh weight compared to time 0. Each column represents data from three independent transgenic lines, ubi::ZmGF14-6 or ZmPR4::ZmGF14-6 lines (15 plants per line). (This figure is available in colour at JXB online.)

Fig. 4.

Expression of drought-associated genes in roots of wild-type (WT) and transgenic lines. Three independent T2 homozygous Ubi::ZmGF14-6 rice plants (lines 4, 9, and 14; 16 plants per line) were grown hydroponically in water for 7 days and then exposed to 20% PEG-8000 (drought) or water (control) for 24 h. qRT-PCR was performed using 2 μg of total RNA with specific primers for (A) rab21 (Os11g26750), (B) dip1 (Os02g44870), and (C) OSE (Os01g64730). Relative expression levels were calculated and normalized with respect to the rice ubiquitin mRNA (OsUbi1, Os06g46770). Each column represents an average of three replicates and bars indicate SDs.

Fig. 5.

Susceptibility or rice plants expressing the ZmGF14-6 gene to infection by F. verticillioides. Three independently T2 homozygous lines expressing ZmGF14-6 under the control of the constitutive promoter (Ubi::ZmGF14-6 lines 4, 9, and 14) or the pathogen-inducible ZmPR4 promoter (ZmPR4::ZmGF14-6 lines 1, 8, and 10) were assayed in disease-resistance assays. Representative results obtained for one line for each transformation event are shown (similar results were observed with the three independent ZmGF14-6 lines for each transformation event). (A) Seeds were germinated for 24 h, inoculated with 50 μl of sterile water (Control, left) or 50 μl of fungal spores (10² spores ml⁻¹) (F. verticillioides, right), and then allowed to continue germination. Picture was taken 10 days after inoculation. Pictures shown are from one of three experiments that gave similar results. (B) Percentage of seeds that overcome infection by the fungus F. verticillioides relative to non-infected seeds. Seedling survival was scored for each line at 10 days after inoculation with fungal spores (the histograms show the mean ± SD from three independent replicates). (This figure is available in colour at JXB online.)

Fig. 6.

Susceptibility of rice plants expressing the ZmGF14-6 gene to infection by M. oryzae. Disease resistance was tested in transgenic lines expressing the ZmGF14-6 gene under the control of either the constitutive ubiquitin (ubi) promoter or the pathogen-inducible ZmPR4 promoter using the detached-leaf assay (Coca et al., 2004). In each case, three independent transgenic lines were assayed with similar results (results for only two of these lines are shown). Reference cultivars used in this study were Maratelli (susceptible) and Saber (resistant). (A) Leaves were locally inoculated with a M. oryzae spore suspension (10⁶ spores ml⁻¹). Disease symptoms 4 days after inoculation of leaves are shown. Results shown are from one of four experiments that produced similar results. (B) Size of lesions (in cm²) produced on the leaves of the control and transgenic ZmGF14-6 rice lines 4 days after inoculation with M. oryzae spores (10⁶ spores ml⁻¹). The average lesion was determined from spot infections by using the APS Assess 2.0 programe (three independent transgenic lines, 12 plants per line, and five inoculations per leaf). Error bars represent SDs. (This figure is available in colour at JXB online.)

Fig. 7.

Expression of the rice defence genes PBZ1 (A) and PR5 (B) in ZmGF14-6 and wild-type (WT) rice plants. Expression analyses were carried out by qRT-PCR in 24 h M. oryzae-infected and non-infected leaves using specific primers for the PBZ1 (Os12g36880) and PR5 (Os3g46070) rice genes. Each RNA was prepared from a pool of leaves from eight plants at the three-leaf stage. Relative expression levels were calculated and normalized with respect to the rice ubiquitin mRNA (OsUbi1, Os06g46770). Results for Ubi::ZmGF14-6 (lines 4 and 9) and ZmPR4::ZmGF14-6 (lines 1 and 8) are shown.

Fig. 8.

Subcellular localization of the ZmGF14-6 protein in onion cells. Onion cells were transformed with ZmGF14-6::GFP (A–C, I–L), GFP (D), or ZmGF14-6::CFP (E–H) via particle bombardment. Confocal images were taken 24 h post-bombardment. Projection images (A, D) and individual sections (B, C, E–L) are shown. Scale bars = 20 μm. (A–C) Cell transformed with ZmGF14-6::GFP. A magnification image of a ZmGF14-6-expressing onion cell is shown in (B) and (C). Fluorescence and transmission images are shown. (D) Onion epithelial cell expressing GFP. (E–H) Co-expression of ZmGF14-6::CFP and VAMP727::YFP, a marker of early endosomes. CFP fluorescence image (E) and YFP fluorescence image (F) are merged in (G). Transmission image is shown in (H). (I–L) Onion epithelial cell expressing ZmGF14-6::GFP were labelled with FM4-64. GFP fluorescence image (I) and FM4-64 fluorescence image (J) are merged in (K). (L) Transmission image of the onion cell.