Callose synthesis occurs at specific stages of cell wall development in all cell types, and in response to pathogen attack, wounding and physiological stresses. We isolated promoters of 12 Arabidopsis callose synthase (CalS1-12) genes and demonstrated that different callose synthases are expressed specifically in different tissues during plant development. That multiple CalS genes are expressed in the same cell type suggests the possibility that CalS complex may be constituted by heteromeric subunits. Five CalS genes were induced by pathogen (Peronospora parasitica, a causal agent of downy mildew) or salicylic acid (SA) treatments, while seven CalS genes were not affected by these treatments. Among the genes that are induced, CalS1 and CalS12, showed the highest responses. When expressed in npr1, a mutant impaired in the response of pathogen related (PR) genes to SA, the induction of CalS1 and CalS12 genes by the SA or pathogen treatments was significantly reduced. The patterns of expression of the other three CalS genes were not changed significantly in the npr1 mutant. These results suggest that the high induction observed of CalS1 and CalS12 is NPR1-dependent while the weak induction of all five CalS genes is NPR1-independent. In a T-DNA knockout mutant of CalS12, callose encasement around the haustoria on the infected leaves was reduced and the mutant was found to be more resistant to downy mildew as compared to the wild type plants. Arabidopsis contains 12 callose synthase (CalS) genes that have evolved in order to catalyze callose synthesis in different locations and in response to biotic and abiotic cues. We demonstrate that one of these genes, CalS5 is responsible for the synthesis of callose deposited to the primary callose wall of meiocytes, tetrads and microspores, and is essential for the exine formation and pollen viability. CalS5 encodes a transmembrane protein of 1923 amino acid residues with a molecular mass of 220 kD. Knockout of the CalS5 gene by T-DNA insertion resulted in a severe reduction in fertility. The reduced fertility in cals5 mutants was attributed to the degeneration of microspores. However, megagametogenesis is not affected and the female gametes are completely fertile in cals5 mutants. CalS5 gene is expressed in several organs with the highest expression in meiocytes, tetrads, microspores and mature pollens. Callose deposition in these tissues in cals5 mutants was nearly completely depleted, suggesting that this gene is essential for the synthesis of callose in these tissues. The pollen exine wall was not formed properly in the mutant and tryphine appeared to be transported from the pollen outer wall into the central vacuole presumably via endocytosis. These data suggest that callose synthesis has a vital function in building the exine sculpture, integrity of which is essential for pollen viability. Using the cell plate specific CalS1 as a bait to screen an Arabidopsis cDNA library constructed in the yeast two-hybrid vector, we obtained two positive clones. One of these interacting clones, RLK1, encodes protein kinase and may play a role in the regulation of CalS1 activity during cell plate formation. Another clone, UGP1, encodes an UDP-glucose pyrophosphorylase and may act to provide alternative source of UDP-glucose for the synthesis of callose. In summary, induction of callose synthase genes by pathogen infection or SA treatment involves both npr1-dependent and npr1-independent signaling pathways. CalS5 is required for exine formation during microgametogenesis and pollen viability in Arabidopsis. UDP-Glc Pyrophosphorylase could be a component of callose synthase complex.