Invasive hyphal growth but not appressorium formation, penetration or IH elaboration is restricted in Δ<i>str3</i> strains.

<p>(<b>A</b>) Live-cell-imaging of rice leaf sheaths, 48 hpi, show that both Guy11 and Δ<i>str3</i> strains can elaborate IH in infected cells. Ap is appressorium on the surface of the leaf and its position is indicated to demonstrate that IH growth occurs in the plant cell and not epiphytically on the surface of the leaf. Scale bar is 10 µm. (<b>B</b>) The mean rate of appressorium formation on the rice leaf surface was determined using the rice leaf sheath assay. 5×10⁴ spores ml⁻¹ of each strain were inoculated onto rice cuticles as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047392#s4" target="_blank">Materials and Methods</a>. At 36 hpi, images were taken, and the number of appressoria that had developed from 50 conidia of each strain was analyzed. This was repeated three times for each strain to determine the mean value. Error bars are standard deviation. Closed bars are values for Guy11 and grey bars are values for Δ<i>str3</i> strains. Bars with the same letter are not significantly different (<i>Student's t-test</i> p≤0.05). (<b>C</b>) Rate of appressorium penetration. The number of appressoria that had penetrated the leaf cuticle and developed visible IH, from a total of 50 appressoria observed, was determined at 36 hpi. The average was determined from three independent replicates. Error bars are standard deviation. Closed bars are values for Guy11 and grey bars are values for Δ<i>str3</i> strains. Bars with the same letter are not significantly different (<i>Student′s t-test</i> p≤0.05). (<b>D</b>) The thickness of IH for each strain was measured. Three leaf sheaths were inoculated with each strain, and 50 IH widths, arising from 50 individual appressoria on the surface, were determined for each replicate at 48 hpi to generate a mean IH width value. Error bars are standard deviation. Closed bars are values for Guy11 and grey bars are values for Δ<i>str3</i> strains. Bars with the same letter are not significantly different (<i>Student's t-test</i> p≤0.05). (<b>E</b>) IH growth was determined for each strain, at 48 hpi, using the four-point scale developed by Saitoh et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047392#pone.0047392-Saitoh1" target="_blank">[24]</a>. According to this scale, 1 = IH length shorter than 10 µm with no branching; 2 = IH length is 10–20 µm with 0–2 branches; 3 = IH length is longer than 20 µm and/or with more than 2 branches within one cell; 4 = IH has spread to adjacent cells. Three leaf sheaths were inoculated with each strain, and 50 IH growth rates were determined for each replicate to generate a value for the mean IH growth rate. Error bars are standard deviation. Closed bars are values for Guy11 and grey bars are values for Δ<i>str3</i> strains. Bars with the same letter are not significantly different (<i>Student′s t-test</i> p≤0.05). (<b>F</b>) IH movement to adjacent cells is curtailed in Δ<i>str3</i> strains. Three leaf sheaths were inoculated with each strain, and the movement of IH into adjacent cells at 48 hpi was observed from a total of 50 primary infected cells per replicate to generate a value for the mean proportion of IH that had moved to adjacent cells (growth level 4). Error bars are standard deviation. Closed bars are values for Guy11 and grey bars are values for Δ<i>str3</i> strains. Bars with the same letter are not significantly different (<i>Student's t-test</i> p≤0.05).