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, 16 (8), 2217-32

Vascular Associated death1, a Novel GRAM Domain-Containing Protein, Is a Regulator of Cell Death and Defense Responses in Vascular Tissues

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Vascular Associated death1, a Novel GRAM Domain-Containing Protein, Is a Regulator of Cell Death and Defense Responses in Vascular Tissues

Séverine Lorrain et al. Plant Cell.

Abstract

The hypersensitive response (HR) is a programmed cell death that is commonly associated with plant disease resistance. A novel lesion mimic mutant, vad1 (for vascular associated death1), that exhibits light conditional appearance of propagative HR-like lesions along the vascular system was identified. Lesion formation is associated with expression of defense genes, production of high levels of salicylic acid (SA), and increased resistance to virulent and avirulent strains of Pseudomonas syringae pv tomato. Analyses of the progeny from crosses between vad1 plants and either nahG transgenic plants, sid1, nonexpressor of PR1 (npr1), enhanced disease susceptibility1 (eds1), or non-race specific disease resistance1 (ndr1) mutants, revealed the vad1 cell death phenotype to be dependent on SA biosynthesis but NPR1 independent; in addition, both EDS1 and NDR1 are necessary for the proper timing and amplification of cell death as well as for increased resistance to Pseudomonas strains. VAD1 encodes a novel putative membrane-associated protein containing a GRAM domain, a lipid or protein binding signaling domain, and is expressed in response to pathogen infection at the vicinity of the hypersensitive lesions. VAD1 might thus represent a new potential function in cell death control associated with cells in the vicinity of vascular bundles.

Figures

Figure 1.
Figure 1.
Phenotype of the vad1 Mutant. (A) and (B) Five-week-old plants of vad1 in comparison with the wild-type plant (Ws-4) (photographed at the same distance) grown under normal light intensity (A) and under low light conditions (B). (C) Spontaneous lesion formation in vad1 plants. Leaves show lesions propagating along the vascular system starting at the petiole basis. (D) Evans blue staining reveals regions of intensely stained dead cells along the vascular system of vad1 leaves. (E) Observation of vad1 leaves after bleaching, under a stereomicroscope, showing the lesions along the vascular system. Bar = 2 mm. (F) Observation of the same leaf as in (E) under a fluorescence microscope (Leica MZ FLIII) shows autofluorescence associated to the lesions observed in (E) (excitation filter 470/40 nm, barrier filter 515 nm). Bar = 2 mm. (G) H2O2 staining of the same leaf using H2DCFDA reveals production of this molecule at the site of lesion formation (excitation filter 470/40 nm, barrier filter 515 nm). Bar = 2 mm. (H) to (K) Microscopic analysis of leaf sections of vad1 under lesion-promoting conditions ([H] and [J]) and of the wild type (Ws-4), healthy (K) and after inoculation with an avirulent strain of Xanthomonas (Xcc147) (I). vad1 lesions (H) resemble Xcc147-induced HR (I). Xylem vessels in vad1 lesions are occluded by pink-stained material (J) not seen in healthy leaves (K). Bars = 12 μm for (J) and (K) and 6 μm for (H) and (I).
Figure 2.
Figure 2.
SA Levels and Defense Gene Expression in Wild-Type and vad1 Plants. (A) and (B) Total SA levels in wild-type and vad1 plants. Leaves were harvested from plants grown on soil under lesion-promoting conditions (A) 10 d before lesion appearance (white bars), at the lesion appearance (hatched bars), and 5 d after the lesion appearance (black bars) and from plants grown under lesion conditions at the same times (B). SA measurements and standard errors are derived from two replicates. F.W., fresh weight. (C) and (D) Transcript levels of PR1 (Pathogenesis-Related 1), PDF1-2 (Plant Defensin 1-2), PR3, and Athsr3 (Arabidopsis thaliana Hypersensitivity-Related 3) in wild-type and vad1 plants at different times before, during, and after lesion appearance in leaves with or without lesions under lesion-promoting conditions (C). The same analysis was performed in wild-type and vad1 plants under lesion conditions at the same times (D). Transcript levels were determined by gel blot analysis.
Figure 3.
Figure 3.
Lesion Phenotypes and Bacterial Growth in Wild-Type and vad1 Plants after Pathogen Inoculation. Leaves of 5-week-old wild-type and vad1 mutant plants grown under lesion-promoting conditions ([A] and [B]) were infiltrated on both sides of the leaf with suspensions (2.107 colony-forming units [cfu]/mL) of P. syringae pv tomato strain DC3000 expressing avrRpm1 (A) and DC3000 (B). Leaves were photographed 54 h after inoculation and were classified according to the propagation rate of the lesions before inoculation: no lesion (vad1), presence of lesion only halfway up the primary vein (vad1+), and lesions propagating along the whole primary vein (vad1++). In the case of DC3000, inoculations with a lower inoculum (2.105 cfu/mL) were performed for a better observation of the disease phenotypes in the wild-type and vad1+ leaves. All treatments were repeated at least three times with similar results. (C) Growth of P. syringae pv tomato DC3000 and DC3000 expressing avrRpm1 in wild-type and vad1 plants grown under lesion-promoting conditions or lesion conditions. In the case of mutant plants grown under lesion-promoting conditions, bacterial growth was evaluated in the different leaves classified as previously described. Inoculation was performed with a bacterial suspension of 2.105 cfu/mL, and bacterial growth determinations were performed at the times indicated. Mean bacterial densities are shown (three to five replicates with corresponding standard deviations) for one representative experiment from two or three independent experiments performed for each strain.
Figure 4.
Figure 4.
Lesion Phenotypes, Bacterial Growth, and Defense Gene Expression in Wild-Type, Single Mutant (nahG and vad1), and Double Mutant vad1 nahG Plants. Two lines, vad1/nahG 37 and vad1/nahG 55, out of three lines are presented as examples. (A) Five-week-old wild-type, single, or double mutant plants 7 d after lesion formation in vad1. (B) Defense gene expression in wild-type, single, or double mutant plants. Transcript levels of PR-1, PR-3, and PDF1-2 were determined by quantitative PCR in plants grown under lesion-promoting conditions 8 d before (white bars), at day 0 (gray bars), and 7 d (black bars) after lesion formation in vad1. See Methods for further details. This experiment was repeated twice with different sets of plants, and similar results were obtained. F.W., fresh weight. (C) Total SA levels in wild-type, single, or double mutant plants 7 d after lesion formation. The plant material used in (B) was also used for SA measurements. (D) Bacterial growth in wild-type, single, or double mutant plants. Inoculation with P. syringae strain DC3000 and strain DC3000 expressing avrRpm1 was performed with a bacterial suspension of 2.105 cfu/mL, and bacterial growth determinations were performed at the times indicated. Mean bacterial densities are shown (three to five replicates with corresponding standard deviations) for one representative experiment from two or three independent experiments performed for each strain.
Figure 5.
Figure 5.
Lesion Phenotypes and Defense Gene Expression in Wild-Type, Single Mutant (sid1, npr1, and vad1), and Double Mutant vad1 sid1 and vad1 npr1 Plants. Two lines, vad1 sid1 3 and vad1 sid1 10, out of nine lines, and vad1/npr1 1 and vad1/npr1 14, out of 16 lines, are presented as examples. (A) and (D) Five-week-old single or double vad1 sid1 (A) and vad1 npr1 (D) mutant plants 10 d after lesion formation in vad1. (C) and (F) Total SA levels in wild-type, single, or double mutant plants vad1 sid1 (C) and vad1 npr1 (F) after lesion formation in vad1. The plant material used in (B) or (E) was also used for SA measurements. (B) and (E) Defense gene expression in wild-type, single, or double vad1 sid1 (B) and vad1 npr1 (E) mutant plants. Transcript levels of PR-1 and AtbohD were determined by quantitative PCR in plants grown under lesion-promoting conditions 9 d before (white bars) and 6 d (black bars) after lesion formation in vad1 (B). In (E), transcript levels of PR-1 and PDF1-2 were also determined by quantitative PCR in plants grown under lesion-promoting conditions 7 d before (white bars) and 5 d (black bars) after lesion formation in vad1. See Methods for further details. F.W., fresh weight.
Figure 6.
Figure 6.
Cell Death Phenotypes in Wild-Type, Single Mutants (eds1, ndr1, and vad1), and Double Mutant vad1 eds1 and vad1 ndr1 Plants. One line, vad1 eds1 15, out of six lines, and vad1 ndr1 4, out of five lines, are presented as examples. (A) and (B) Single or double mutant plants (vad1 eds1) 5 d (A) and 8 d (B) after lesion formation in vad1. (C) and (D) Single or double mutant (vad1 ndr1) plants 3 d (A) and 11 d (B) after lesion formation in vad1. (E), (H), (G), and (J) Defense gene expression in wild-type, single, or double mutant plants. Transcript levels of PR-1 and PDF1-2 were determined by quantitative PCR in plants grown under lesion-promoting conditions 6 d before (white bars) and 6 d (gray bars) and 12 d (black bars) after lesion formation in vad1 for vad1 eds1 (E) and 6 d before (white bars) and 2 d (gray bars) and 8 d (black bars) after lesion formation in vad1 for vad1 ndr1 lines (F). See Methods for further details. This experiment was repeated twice with different sets of plants, and similar results were obtained. F.W., fresh weight. (F) and (I) Total SA levels in wild-type, single, or double mutant plants vad1 eds1 (F) and vad1 ndr1 (I) at two time points after lesion formation in vad1. The plant material used in (E) or (H) was also used for SA measurements.
Figure 7.
Figure 7.
Resistance Phenotypes in Wild-Type, Single Mutant (eds1, ndr1, and vad1), and Double Mutant vad1 eds1 and vad1 ndr1 Plants. Bacterial growth in wild-type, single, or double mutant plants: vad1 eds1 (A) and vad1 ndr1 (B). Inoculation with P. syringae strain DC3000 and strain DC3000 expressing avrRpm1, avrRpt2, and avrRps4 was performed with a bacterial suspension of 2.105 cfu/mL, and bacterial growth determinations were performed at the times indicated (day 0, white bars; day 3, black bars). Mean bacterial densities are shown (three to five replicates with corresponding standard deviations) for one representative experiment from two or three independent experiments performed for each strain.
Figure 8.
Figure 8.
Molecular Identification of the VAD1 Gene. (A) Genomic organization of vad1. The arrows indicate the insertion sites of the T-DNA in the mutants vad1-1 and vad1-2 within the VAD1 gene sequence. Gene organization in exons (boxes) and introns (black line) is presented. (B) Comparison of the predicted VAD1 protein with a protein from C. elegans (ZC328.3, http://elegans.swmed.edu). The regions delimiting the different domains were deduced from ProDom and SMART analysis.
Figure 9.
Figure 9.
VAD1 Gene Expression after Pathogen Inoculation and in Response to SA Treatment. (A) VAD1 and PR1 transcript accumulation in wild-type plants (Col-0) at different times after inoculation with an avirulent (Xcc147) strain of X. campestris pv campestris (squares), an avirulent (DC3000/avrRpm1) strain of P. syringae pv tomato (diamonds), or after treatment with water (triangles). Transcript levels of VAD1 and PR-1 were determined by quantitative PCR as described in Methods. Results are expressed as fold induction compared with the noninoculated wild type. (B) to (E) Histochemical localization of GUS activity in leaves from vad1 plants or plants containing a VAD1 promoter-GUS fusion, both healthy (B) and after inoculation with an avirulent (Xcc147) of X. campestris pv campestris ([C], [D], and [E]). Undetached leaves were infiltrated in a small region (1 cm2) with the bacterial strain at 108 cfu/mL and observed 48 h postinoculation (C) or sprayed with the bacterial suspension at 108 cfu/mL 6 h postinoculation (D) or 72 h postinoculation (E). (F) VAD1 and PR1 transcript accumulation in wild-type (Ws-4) and npr1 mutant plants at different times after treatment with SA (1 mM). One representative experiment is shown from two independent experiments.

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