To plot the line in the overlay, you can use line()
line([x1 x2], [y1 y2], 'Color', 'r');
To "burn" the line into the image, you can either use normal indexing if the line is along rows and columns
grayImage(row1:row2, col) = 255; % or whatever intensity you want
or you can use imline() if the points are not in the same row or column, as the attached demo illustrates.
