Coordinated regulation of genes for secretion in tobacco at late developmental stages: association with resistance against oomycetes

Abstract

Besides the systemic acquired resistance (SAR) induced in response to microbial stimulation, host plants may also acquire resistance to pathogens in response to endogenous stimuli associated with their own development. In tobacco (Nicotiana tabacum), the vegetative-to-flowering transition comes along with a susceptibility-to-resistance transition to the causal agent of black shank disease, the oomycete Phytophthora parasitica. This resistance affects infection effectiveness and hyphal expansion and is associated with extracellular accumulation of a cytotoxic activity that provokes in vitro cell death of P. parasitica zoospores. As a strategy to determine the extracellular events important for restriction of pathogen growth, we screened the tobacco genome for genes encoding secreted or membrane-bound proteins expressed in leaves of flowering plants. Using a signal sequence trap approach in yeast (Saccharomyces cerevisiae), 298 clones were selected that appear to encode for apoplastic, cell wall, or membrane-bound proteins involved in stress response, in plant defense, or in cell wall modifications. Microarray and northern-blot analyses revealed that, at late developmental stages, leaves were characterized by the coordinate up-regulation of genes involved in SAR and in peroxidative cross-linking of structural proteins to cell wall. This suggests the potential involvement of these genes in extracellular events that govern the expression of developmental resistance. The analysis of the influence of salicylic acid on mRNA accumulation also indicates a more complex network for regulation of gene expression at a later stage of tobacco development than during SAR. Further characterization of these genes will permit the formulation of hypotheses to explain resistance and to establish the connection with development.

Figure 1.

PCA of microarray data. Variables (normalized intensity values or individuals [genes] were plotted separately on graph by report of the first two principal components (PC1 and PC2). A, Distribution of normalized intensity values; C, distribution of genes from water (S) and cryptogein (R)-treated plants. B, Distribution of normalized intensity values; D, distribution of genes from vegetative (S) and flowering plants (R). PCA1 and PCA2 represent 78% and 69% of the total variability in A and C and B and D, respectively.

Figure 2.

Protein gel-blot analysis of extracellular PR proteins of tobacco leaf. Intercellular fluids were prepared from leaves of tobacco plants: lane 1 corresponds to proteins (1 μg) from Xanthi leaves 48 h after stem treatment with cryptogein and SAR induction; lanes 2 and 3 correspond to proteins (25 μg) from leaves of Xanthi plants 50 or 110 d after seeding, respectively.

Figure 3.

Influence of SA accumulation on gene expression during developmental resistance. Each bar represents the values of relative abundance for the indicated gene, between vegetative and flowering Xanthi (white bars) or NAHG-8 (black bars) plants. Values are the means ± sd of two replicates of three different experiments. Differences between Xanthi NAHG-8 plants are statistically significant (P < 0,01, n = 3) for PR-1a and NtTLRP genes.