Functional analysis of effector genes from Xanthomonas species and insights into their evolution in natural populations

The avrXa7 gene from Xanthomonas oryzae pv. oryzae (Xoo) exhibits avirulence and virulence functions in rice. Strains of Xoo that displayed a continuous variation in both of these functions were identified from a field study. To understand the molecular basis for variation in avrXa7 functions, we sequenced the 4.3 kb avrXa7 alleles from seven field strains that exhibited a range in aggressiveness. The avrXa7 gene is one of >12 members of the avrBs3/pthA gene family found in Xoo. The central region of avrXa7 contains 25.5 copies of a 102 bp repeat unit. The 3 '-end encodes structures important for virulence and avirulence, including nuclear localization signal sequences (NLS) and an acidic activation domain (AAD). Four types of avrXa7 mutant alleles were identified with changes ranging from a single base pair change to multiple mutations throughout the alleles; the latter is suggestive of recombination between gene family members. One mutant allele contained an extra repeat unit while another had lost three repeats. In some cases, strains with identical mutant alleles exhibited different levels of aggressiveness to rice, suggesting that second site mutations may account for variation in their phenotypes. Thus, adaptation of Xoo to Xa7 rice fields involves not only alterations at the avrXa7 allele, but may include changes in other gene family members.; Two key structures for virulence and avirulence are located in the 3 '-end of avrBs3/pthA effector gene family members, the NLS and AAD. To identify other regions that are important for these functions, we constructed a series of chimeric genes using two avrBs3/pthA members from X. axonopodis pv. citri (Xac) (apl1 and apl2 genes) and two members from Xoo (avrXa7 and avrXa10). The avrXa7 gene caused a hypersensitive response in citrus, but apl1-containing constructs did not induce any response on rice. A 417 bp HincII-SphI fragment in the 3'-end was found to be essential for avirulence and virulence to both citrus and rice.