The pilA gene, which encodes the major structure of pili, is required for infection of Xanthomonas axonopodis pv. citri (X. a. pv. citri) by the filamentous bacteriophage Cf. Two open reading frames (ORFs) located downstream of pilA were cloned and characterized. One 1392-bp ORF encodes a protein of 464 amino acids which shares substantial similarity with pilR of other bacterial species; the second ORF (orf618), of 1854-bp, shares sequence similarity with pilS. The existence of the pilR-like and pilS-like genes in various X. campestris pathovars indicated that these two genes are well conserved in Xanthomonas. pilR and pilS mutants were constructed by gene replacement. We found that a pilR mutant, resistant to the infection of phage Cf, was unable to synthesize PHA protein; however, the abundance of the PHA protein and of the pilA transcript was markedly increased by the introduction of a plasmid containing the cloned pilR gene. The restoration of the normal pilus-specific sensitivity of this transformed clone to Cf indicated that the pilR gene functions as a transcriptional regulator of pilA. The pilS mutant, however, was susceptible to Cf infection, and the level of pilA expression in this mutant was similar to that of wild-type cells. Promoter analysis of luciferase reporter gene constructs containing the 5' untranslated regions of pilR or pilS genes revealed that, although the pilR and pilS are contiguous in X. a. pv. citri, the two genes are expressed independently, and the strong pilR promoter leads to the accumulation of PilR in X. a. pv. citri, which positively regulates the biosynthesis of PilA. These results revealed the enhanced sensitivity of X. a. pv. citri to phage Cf in the presence of PilR and indicated that the filamentous phage Cf utilize bacterial pili as a receptor site for its infection.