Figure 2.

DNA Replication in M. oryzae Is Necessary for Initiation of Appressorium Development.

(A) The effect of chemical inhibition of DNA replication on appressorium development in M. oryzae. Water (white bars) or the DNA synthesis inhibitor HU (+HU, black bars) was added (200 mM) to conidial suspensions of the H1:RFP strain, 0 or 8 h postinoculation (hpi). The percentage of conidia developing undifferentiated germ tubes (germlings), swollen germ tube tips (initial appressoria), and differentiated infection cells (mature appressoria) was recorded after 8 h.

(B) Temperature sensitivity of the nim1^I327E mutant of M. oryzae; two putative nim1^I327E transformants (2 and 41) and Guy-11 were incubated at a permissive temperature of 24°C or a restrictive temperature of 32°C for 4 d. Restoration of hyphal growth was assessed by incubation for a further 3 d at 24°C.

(C) Representative images of the nim1^I327E mutant during appressorium development, in the presence (bottom panels) or absence (top panels) of HU; nim1^I327E and Guy-11 germinating conidia were fixed and stained with the DNA-specific stain 4',6-diamidino-2-phenylindole 24 h postinoculation, following incubation at 24 or 29°C. Bars = 10 μm.

(D) Bar charts to show frequency of appressorium development in M. oryzae nim1^I327E. Conidial suspensions of nim1^I327E transformants 2 and 41 were incubated on hydrophobic surfaces at 24 or 29°C. The percentage of conidia developing undifferentiated germ tubes (germlings), swollen germ tube tips (initial appressoria), or differentiated infection cells (mature appressoria) was recorded after 24 h.

(E) Bar charts to show the frequency of germinating conidia displaying a premitotic arrest (pre-M arrest; white bars) or the ability to progress into mitosis uninhibited (mitotic entry; black bars).

(F) HU was added to nim1^I327E and Guy-11 conidia at 0 h and nuclear division and appressorium development assessed 24 h later following incubation at 24°C (left bar chart) or 29°C (right bar chart). All graphs represent two biological and three technical replicate observations of 100 germinating conidia. Error bars are 1 se.