Construction of secretion vectors and use of heterologous signal sequences for protein secretion in Clavibacter xyli subsp. cynodontis.

We have constructed a vector to assess the ability of heterologous signal sequences to function in the endophytic bacterium Clavibacter xyli subsp. cynodontis. This secretion reporter contains the phoA gene from Escherichia coli from which the promoter and signal sequences have been deleted. Signal sequences of streptomycete origin were cloned into the vector and the level of secreted alkaline phosphatase determined enzymatically and by Western blotting. We show that a number of the signal sequences of streptomycete origin function well in C. xyli subsp. cynodontis. By inoculating corn plants with C. xyli subsp. cynodontis expressing the sti2-phoA fusion, we demonstrated alkaline phosphatase activity in planta. Our data show that phoA can be used to detect endophytic bacteria in some locations in planta, and is therefore useful in the study of plant-microbe interactions. Furthermore, our data illustrate how phoA fusions can be useful as a reporter for protein secretion activity of C. xyli subsp. cynodontis in planta.