BACKGROUND
the filamentous ascomycete fungus magnaporthe oryzae causes the economically important blast disease on rice and other crops  <cit> .
several key cellular events that occur during rice infection have been documented .
on the rice leaf surface, the fungus forms a specialized infection structure, called an appressorium, which produces a narrow penetration peg to breach the plant surface  <cit> .
upon penetration, the peg expands to form a filamentous primary hypha that grows into the rice cell and subsequently differentiates into bulbous invasive hyphae  with a pseudohyphal appearance due to regular constrictions at presumed septa  <cit> .
cell cycle regulation is important for the appressorium development and ih proliferation .
semi-closed mitosis has been proposed to occur in m. oryzae, during which the nuclear envelope remains intact, but nuclear pore complexes partially dissociate in the appressorium and ih  <cit> .
the primary hypha and ih invade living host cells  while contained within a host-derived extra-invasive hyphal membrane   <cit> .
the ih grow throughout the first invaded cell before invading adjacent cells  <cit>  .
the ih colonize each new cell biotrophically, but eventually kill host cells ; the invaded cells lose viability by the time the ih move into adjacent cells  <cit> .fig.
<dig> primary hypha differentiates bulbous growth prior to mitosis.
a schematic diagram of ih development inside rice cells , seen from above.
left : a filamentous primary hypha , derived from the appressorium  on the rice cell surface, grows inside the rice cell.
the hyphal apex is associated with the tip bic .
middle : the primary hypha differentiates into bulbous ih, and the tip bic becomes the side bic .
right : ih have filled the first invaded rice cell, and ih with secondary bics are growing into a neighboring cell.
b hyphal development of m. oryzae ckf <dig> in yt <dig> rice sheath cells at 26– <dig> hpi.
shown are maximum intensity projections of confocal optical sections .
merged fluorescence shows fungal cytoplasmic eyfp  and nuclear tdtomato .
top: the appressorium  contained one nucleus  and produced an anucleate filamentous primary hypha at  <dig> hpi.
middle: the tip of the primary hypha swelled but remained anucleate at  <dig> hpi.
bottom: the bulbous end of the primary hypha had become nucleated at  <dig> hpi.
arrows indicate the apical region of the primary hypha which correlates with the hyphal diameter measurements in .
bar = 5 μm.
c graph showing measurements of the diameter at the apical region of the primary hypha, corresponding to the location of arrows in .
groups of infection sites were plotted on the y-axis in 1 μm hyphal diameter increments and the x-axis shows number of infection sites .
anucleate hypha and nucleated hypha are differentiated by blue and red bars, respectively
during the biotrophic invasion, the primary hypha and ih secrete both cytoplasmic effector proteins  that enter the host cytoplasm and apoplastic effectors  that are retained in the eihm compartment .
these effectors are known or presumed to suppress host defense and facilitate infection  <cit> .
cytoplasmic effectors are preferentially localized in the biotrophic interfacial complex , which has been hypothesized as the site of effector translocation into the host cytoplasm  <cit> .
two stages of bics have been previously described  <cit>  .
the tip bic is associated with the apex of the primary hypha.
as the hypha develops, the bic is positioned on the side of the first ih cell, and thus it is called the side bic.
the first ih cell, also known as the side bic-associated cell, is a specialized effector-secreting cell essential for a successful infection.
giraldo et al.
have shown that this ih cell uses two distinct pathways for effector secretion.
one is the conventional secretory pathway for secretion of apoplastic effectors into the eihm compartment; the other is the exocyst components exo <dig> and sec5-involved pathway for secretion of cytoplasmic effectors into the bic.
in addition, this first ih cell is critical in its role as the mother cell of the subsequent biotrophic ih cells.
although our knowledge of early biotrophic invasion has increased, questions remain to be answered, such as how the first ih cell develops inside live host cells, what mechanisms control hyphal differentiation, and what cellular processes are involved in bic development and effector trafficking.
in this study, we provide insights into some of these questions by using live-cell imaging of m. oryzae invasion of susceptible rice cells.
our studies reveal a series of cellular events associated with the first ih cell development.
these studies lead to new mechanistic questions concerning in planta hyphal differentiation and effector trafficking.
RESULTS
apical swelling of the primary hypha precedes mitosis
we generated m. oryzae transformants constitutively expressing eyfp  and histone h1-tdtomato fusion protein .
these transformants exhibited wild-type morphology and pathogenicity .
to characterize the development of the first ih cell upon host penetration , we used one of these transformants, ckf <dig>  and imaged  <dig> random infection sites from  <dig> separate inoculations and microscopy sessions at 22– <dig> h post inoculation .
quantitative analysis of these images revealed three sequential growth stages :  the filamentous primary hypha with one nucleus in the appressorium,  the apically bulbous primary hypha with one nucleus in the appressorium, and  the apically bulbous primary hypha that contains a nucleus with another nucleus in the appressorium.
we quantified these observations by measuring the diameter of the primary hypha close to the appressorium and the diameter at the apical region of the primary hypha before or after swelling .
at all growth stages, the diameter of the primary hypha close to the appressorium was conserved with a mean of  <dig>  μm .
in infection sites with the anucleate primary hypha and a single nucleus present in the appressorium, the diameter of the primary hypha tip had a mean of  <dig>  μm .
in infection sites displaying one nucleus in the appressorium and another one in the bulbous portion of the primary hypha, the diameter of the bulbous portion had a mean of  <dig>  μm , frequently ranging from  <dig>  to  <dig>  μm.
taken together, these data indicate that nuclear division and migration typically occur after the apical tip of the primary hypha has swollen to over 4 μm in diameter.
mitosis in the appressorium and subsequent migration of a single nucleus into the first bulbous ih cell
to determine nuclear dynamics during development of the first ih cell, we conducted time-lapse microscopy of rice cells invaded by m. oryzae mitotic reporter strain ckf <dig> expressing h1-tdtomato and gfp-nuclear localization signal   <cit> .
consistent with the previous study  <cit> , after plant penetration, nuclear division occurred in the appressorium, and subsequently one mitotic nucleus migrated through the primary hypha to arrive in the swollen portion of the primary hypha, while the other nucleus remained in the appressorium .
the timing of nuclear division was consistent with our prediction that nuclear division occurs in infections that had a swollen primary hypha tip lacking a nucleus .
intriguingly, the nucleus that remained in the appressorium appeared to associate with the penetration pore as the other nucleus migrated through the primary hypha to the swollen hypha tip .
our observation of nuclear division within the appressorium contrasts a previous report, in which the appressorial nucleus migrates to the primary hypha and then undergoes mitosis  <cit> .
we considered the possibility that the discrepancy might have arisen because different strains of m. oryzae and rice were used, specifically m. oryzae o-137-derived strain  infecting rice strain yt <dig> in our study and m. oryzae guy11-derived strain  infecting rice strain co- <dig> in the other study  <cit> .
however, we ruled out this possibility because we observed the consistent pattern of nucleus division within the appressorium followed by migration of a single nucleus into the primary hypha when m. oryzae guy11-derived strain  infects yt <dig>  or co- <dig> .
the reason for this discrepancy remains to be determined.
it is worth noting, however, that our findings are consistent with colletotrichum gloeosporioides, in which nuclear division occurs in the appressorium, and then one nucleus migrates into the penetration hypha, whereas the other nucleus remains in the appressorium  <cit> .fig.
<dig> nuclear division occurs within the appressorium.
a
m. oryzae ckf <dig>  infecting rice cultivar yt <dig>  shown are single plane confocal images of both red fluorescence and bright-field , and red fluorescence alone .
nuclei are denoted by arrowheads.
a single nucleus tagged with tdtomato  separated into two mitotic nuclei within the appressorium.
one nucleus then migrated out of focus into the primary hypha, while the other remained in the appressorium.
b
m. oryzae guy11-derived transformant infecting yt <dig> at the same growth stage as .
nuclear dynamics were consistent with , with nuclear division occurring within the appressorium  and migration of a single nucleus out of focus into the primary hypha.
c
m. oryzae guy11-derived transformant infecting rice cultivar co- <dig> at the same growth stage as both  and .
nuclear dynamics were again consistent with nuclear division occurring in the appressorium  and migration of one nucleus into the primary hypha.
bar = 5 μm
formation of the first septum after nuclear division and migration
to define the location and timing of septation during development of the first ih cell, we generated m. oryzae transformants expressing fluorescently labeled septa together with h1-tdtomato.
the septa were visualized by constitutively expressing the plasma membrane-targeted plc delta:ph as a translational fusion to gfp  in m. oryzae.
plc delta:ph binds to pip <dig> lipids  <cit>  and has been shown to localize to the plasma membrane and septa in neurospora crassa  <cit> .
m. oryzae transformants expressing gfp-plc delta:ph showed green fluorescence localized at the plasma membrane of the appressorium and primary hypha .
after nuclear division, one nucleus moved to the bulbous tip of the primary hypha, and the other nucleus remained in the appressorium .
subsequently, the green fluorescence localized near the midpoint of the filamentous portion of the primary hypha, indicating the site of the first septum .
the primary hypha was previously characterized by heath et al.
and kankanala et al.
: it is a filamentous structure that differentiates from the penetration peg, an extremely narrow structure that originates at the appressorium and forces through the plant cell wall, and that subsequently differentiates into bulbous ih.
our septation result further reveals that the primary hypha is a filamentous part of two distinct cell types, constituting the invasive extension of the single-celled appressorium and the first ih cell.fig.
<dig> the first post-penetration septum forms in the primary hypha.
shown are maximum intensity projections of confocal optical sections of a rice cell invaded by m. oryzae strain ckf <dig> at  <dig> hpi.
merged nuclear tdtomato  and gfp fused to plcdelta:ph  are shown.
top: one nucleus  was located in the appressorium  and gfp-plcdelta:ph was localized to the plasma membrane.
middle: the same infection site imaged 50 min later showing one appressorial nucleus and one nucleus in the bulbous hypha.
bottom: the same infection site after an additional 25 min showing gfp fluorescence concentrating at the location of the first septum .
bar = 5 μm
development of bics during hyphal differentiation
two-stage development of bics has been previously documented  <cit>  : the tip bic is associated with the apex of the primary hypha; as the primary hypha switches to bulbous ih growth, the bic maintains its position on what is now the side of the bulbous ih.
to determine how the tip bic becomes the side bic, we used our earlier observations about the time of nuclear division relative to hyphal swelling  to design an experiment.
we imaged m. oryzae transformant ckf <dig> expressing cytoplasmic eyfp and nuclear mrfp as well as mrfp fused to the bic-localized cytoplasmic effector pwl <dig>  <cit>  at different growth stages analogous to the three panels in fig.
1b.
in cases of no bulbous differentiation, the bic was at the tip of the primary hypha .
when bulbous differentiation had occurred, but nuclear division had not, the bic was already at the side, indicating that the bic is displaced from its tip position as the primary hypha swells .
in infections where nuclear division and migration had taken place, we observed the bic still at the side, where it remained as a new hypha began to grow at the tip .
these results indicate that the bulbous swelling of the primary hypha occurs prior to nuclear division and is responsible for the bic being “left” on the side of the cell.
this displacement of the bic indicates that there is depolarization at the apex of the primary hypha during bulbous differentiation.
the position of the bic on one side of the bic-associated cell suggests that the opposite side of the primary hypha grows more than the bic side.
this asymmetric swelling produces an oval cell offset from the center of the hypha  rather than a round cell, which would result from equal swelling in all directions.
this growth is clearly distinct from the polarized growth observed during filamentous primary hypha development, of which the tip bic is characteristic.
polarity in filamentous fungi is established and maintained by multiple proteins, especially those associated with control of cytoskeletal positioning  <cit> .
the spitzenkörper  and polarisome have been established as instrumental in determining the direction of hyphal tip growth in filamentous fungi .
in m. oryzae, spk-associated motor protein mlc <dig> is localized to the growing tip of the primary hypha before differentiation of the bic-associated cell  <cit> .
after differentiation, mlc <dig> is not present at the growing ih tip, but can be visualized near septa and in the subapical bic-associated cell  <cit> .
in contrast, the polarisome component spa <dig> is localized to the growing tip of the primary hypha and ih  <cit> .
because an ih cell develops from the growing end of the bic-associated cell, there must be a repolarization event after asymmetric swelling occurs.
the spa <dig> and other polarisome proteins are likely involved in repolarization of the ih growing tip.fig.
<dig> bic development.
a tip to side bic differentiation and first nuclear division event at  <dig> hpi.
shown are maximum intensity projections of confocal optical sections .
images are of m. oryzae ckf <dig>  expressing cytoplasmic eyfp , nuclear mrfp , and bic-localized mrfp .
arrows = bics.
arrowheads = nuclei.
asterisk = appressorium.
left: tip bic stage.
middle: side bic stage.
right: after nuclear division, the side bic remained at the same position.
bars = 5 μm.
b representative measurement from the penetration point to the side bic in m. oryzae ckf <dig> at  <dig> hpi.
images show bic-localized mcherry , fungal hypha outlined by egfp , and mcherry and egfp overlapping in the side bic .
top: a maximum intensity projection of confocal optical sections  and 2-dimensional length  measured with the open bezier tool in the zen black software.
bottom: a 3-dimensional reconstruction of the same infection site shows 3d length  measured with the imaris dendrite creation algorithm .
bars = 5 μm.
c comparative graphical summary of measurements taken with 3d imaris and with 2d zen black software of the distance between the penetration point to the side bic.
individual infection sites  have both a 3d measurement  and a corresponding 2d measurement .
the vertical distance between a data point and the 1-to- <dig> line  on the graph corresponds to the difference in length between the 2d and 3d measurements associated with that data point
regular growth from the appressorium penetration to the side bic
the filamentous primary hypha appeared to extend from the appressorium to a consistent distance before widening at its apical region.
we reasoned that the side bic would be positioned at a defined distance from the appressorium.
to test this, we measured the distance from the appressorium penetration point to the side bic using m. oryzae transformant ckf <dig>  expressing the bic-localized cytoplasmic effector pwl <dig> fused to mcherry:nls and also ih-outlining apoplastic effector bas <dig> fused to egfp  <cit> .
the penetration point was identified by examining the egfp fluorescence outlining the primary hypha in conjunction with the bright-field, and the bic was identified based on the strongly localized mcherry fluorescence.
we conducted two-dimensional measurements of these images using the zen black open bezier tool .
the distance along the primary hypha from the penetration point to the side bic has an average of 36 μm, ranging from  <dig> to 45 μm  .
to obtain more accurate measurements of the same infection sites, we also used the three-dimensional dendrite measurement algorithm in imaris .
an additional movie file shows the three-dimensional measurement in more detail .
the average distance was 43 μm, ranging from  <dig> to 54 μm  .
our finding of this considerable distance raises an intriguing question concerning effector secretion.
cytoplasmic effectors such as pwl <dig> are secreted into the tip bic and the side bic, which are located more than 32 μm from the appressorial nucleus  in which the pwl <dig> gene is expressed .
there must be a mechanism to transport effectors from the appressorium through the primary hypha for their secretion to the distantly located bic, but the mechanism remains to be determined.
additional file 2: movie s <dig>  three-dimensional measurement from the appressorium penetration point to the bic of m. oryzae strain ckf <dig> growing in a rice cell at  <dig> hpi.
apoplastic effector bas <dig> with a translational fusion to egfp  outlined the invasive hypha.
the bic and the rice nucleus were visualized by bic-accumulating cytoplasmic effector pwl <dig> with a translational fusion to mcherry:nls .
overlap of green and red signals is shown in yellow.
source image is a series of 1 μm confocal optical sections taken over a depth of 23 μm.
movie was created using the key frame animation feature in imaris software .
at the start of the animation, the penetration point is on the left, and the bic is on the right.
as the animation rotates, a penetration point-to-bic measurement  is overlaid on the image.
the measurement was performed with the dendrite creation algorithm in the imaris software  and has a length of  <dig>  μm.
a maximum intensity projection of the same infection site in this movie is shown in fig.
4b , and a mirrored still of the last frame in this movie is shown in fig.
4b .
bar = 5 μm.
hyphal growth inside heat-killed rice cells
we hypothesized that the development we observed during the early biotrophic invasion is specific to live host cells.
to test this, we used a heat-killed cell assay, inoculating nonviable rice tissue with conidia.
when inoculated on this tissue, m. oryzae produced the normal appressorium , indicating that the surface cues were still sufficient for appressorium differentiation.
after penetration into heat-killed host cells, the fungus did not show distinct differentiation from filamentous to bulbous growth as observed during invasion of live host cells, but elongated, filamentous-like growth throughout the rice cell and its neighbors .
hyphae growing in heat-killed cells spread rapidly into adjacent cells rather than completely filling the first cell, which the ih in living cells do over the course of 12 h  <cit> .
in living host cells, bics are easily recognized in bright-field images due to their predictable locations on the tip of the primary hypha, the side of the first bulbous ih cell, and the tips and sides of hyphae moving into adjacent host cells  <cit>  .
we did not observe any indications of bics in the bright-field images of infections in heat-killed cells.
the absence of bics in dead host cells is consistent with giraldo et al.
showing that bics are derived from host membrane components such as the plasma membrane and endoplasmic reticulum; therefore, bics are unlikely to form in host cells, which lack intact membranes.fig.
<dig> hyphal morphology in heat-killed rice cells is distinct from growth in live cells.
m. oryzae ckf <dig>  expressing cytoplasmic eyfp  and nuclear tdtomato , growth in heat-killed rice cells at  <dig> hpi.
shown are merged fluorescence confocal images of a single focal plane , or a projection   of the same infection site.
rice cell walls are shown as white lines.
top: a single focal plane shows the appressorium  with a nucleus, germ tube , and hypha  that grew on the rice surface.
middle: a focal plane 12 μm beneath the appressorium shows  <dig> filamentous-like ih branches  and locations where ih had crossed the rice cell wall into neighboring cells .
bottom: a maximum intensity projection shows all features of both focal planes .
bar = 10 μm
CONCLUSIONS
we provide cellular details of early post-penetration development of m. oryzae during invasion of susceptible rice cells.
this study defines developmental stages, in which the single-celled appressorium gives rise to the first ih cell, and the tip bic becomes the side bic when the tip bic-associated hyphal apex undergoes asymmetric swelling prior to mitosis in the appressorium .
development of bics and bic-associated cells is induced during invasion of live rice cells.
our study provides a descriptive framework for early biotrophic invasion of rice leaf sheath tissue.
it remains to be determined whether the cellular processes reported in this study are conserved during infection of other rice tissues and of other plant species.
this study also leads to new mechanistic questions concerning effector trafficking from the appressorium to the bic and hyphal differentiation in response to host environmental cues.fig.
<dig> schematic diagram of early post-penetration growth in susceptible rice cells.
pre-penetration: m. oryzae conidium  germinates and forms an appressorium  containing one nucleus .
post-penetration:  the appressorium penetrates the host cell wall, forming an anucleate filamentous primary hypha  with the tip bic .
the tip of the filamentous primary hypha swells and the tip bic becomes the side bic .
the primary hypha remains anucleate.
nuclear division occurs in the appressorium.
one mitotic nucleus migrates into the bulbous portion of the primary hyphal tip, while the other nucleus remains in the appressorium.
the first septum forms in the filamentous portion of the primary hypha , finalizing the establishment of the effector-secreting bic-associated cell.
polarized growth resumes from the bic-associated cell

 the filamentous ascomycete fungus magnaporthe oryzae causes the economically important blast disease on rice and other crops. several key cellular events that occur during rice infection have been documented. on the rice leaf surface, the fungus forms a specialized infection structure, called an appressorium, which produces a narrow penetration peg to breach the plant surface. upon penetration, the peg expands to form a filamentous primary hypha that grows into the rice cell and subsequently differentiates into bulbous invasive hyphae with a pseudohyphal appearance due to regular constrictions at presumed septa. cell cycle regulation is important for the appressorium development and ih proliferation. semi-closed mitosis has been proposed to occur in m. oryzae, during which the nuclear envelope remains intact, but nuclear pore complexes partially dissociate in the appressorium and ih. the primary hypha and ih invade living host cells while contained within a host-derived extra-invasive hyphal membrane. the ih grow throughout the first invaded cell before invading adjacent cells. the ih colonize each new cell biotrophically, but eventually kill host cells; the invaded cells lose viability by the time the ih move into adjacent cells.fig. primary hypha differentiates bulbous growth prior to mitosis. a schematic diagram of ih development inside rice cells, seen from above. left: a filamentous primary hypha, derived from the appressorium on the rice cell surface, grows inside the rice cell. the hyphal apex is associated with the tip bic. middle: the primary hypha differentiates into bulbous ih, and the tip bic becomes the side bic. right: ih have filled the first invaded rice cell, and ih with secondary bics are growing into a neighboring cell. b hyphal development of m. oryzae ckf in yt rice sheath cells at 26– hpi. shown are maximum intensity projections of confocal optical sections. merged fluorescence shows fungal cytoplasmic eyfp and nuclear tdtomato. top: the appressorium contained one nucleus and produced an anucleate filamentous primary hypha at hpi. middle: the tip of the primary hypha swelled but remained anucleate at hpi. bottom: the bulbous end of the primary hypha had become nucleated at hpi. arrows indicate the apical region of the primary hypha which correlates with the hyphal diameter measurements in. bar = 5 μm. c graph showing measurements of the diameter at the apical region of the primary hypha, corresponding to the location of arrows in. groups of infection sites were plotted on the y-axis in 1 μm hyphal diameter increments and the x-axis shows number of infection sites. anucleate hypha and nucleated hypha are differentiated by blue and red bars, respectively during the biotrophic invasion, the primary hypha and ih secrete both cytoplasmic effector proteins that enter the host cytoplasm and apoplastic effectors that are retained in the eihm compartment. these effectors are known or presumed to suppress host defense and facilitate infection. cytoplasmic effectors are preferentially localized in the biotrophic interfacial complex, which has been hypothesized as the site of effector translocation into the host cytoplasm. two stages of bics have been previously described. the tip bic is associated with the apex of the primary hypha. as the hypha develops, the bic is positioned on the side of the first ih cell, and thus it is called the side bic. the first ih cell, also known as the side bic-associated cell, is a specialized effector-secreting cell essential for a successful infection. giraldo et al. have shown that this ih cell uses two distinct pathways for effector secretion. one is the conventional secretory pathway for secretion of apoplastic effectors into the eihm compartment; the other is the exocyst components exo and sec5-involved pathway for secretion of cytoplasmic effectors into the bic. in addition, this first ih cell is critical in its role as the mother cell of the subsequent biotrophic ih cells. although our knowledge of early biotrophic invasion has increased, questions remain to be answered, such as how the first ih cell develops inside live host cells, what mechanisms control hyphal differentiation, and what cellular processes are involved in bic development and effector trafficking. in this study, we provide insights into some of these questions by using live-cell imaging of m. oryzae invasion of susceptible rice cells. our studies reveal a series of cellular events associated with the first ih cell development. these studies lead to new mechanistic questions concerning in planta hyphal differentiation and effector trafficking.  apical swelling of the primary hypha precedes mitosis we generated m. oryzae transformants constitutively expressing eyfp and histone h1-tdtomato fusion protein. these transformants exhibited wild-type morphology and pathogenicity. to characterize the development of the first ih cell upon host penetration, we used one of these transformants, ckf and imaged random infection sites from separate inoculations and microscopy sessions at 22– h post inoculation. quantitative analysis of these images revealed three sequential growth stages: the filamentous primary hypha with one nucleus in the appressorium, the apically bulbous primary hypha with one nucleus in the appressorium, and the apically bulbous primary hypha that contains a nucleus with another nucleus in the appressorium. we quantified these observations by measuring the diameter of the primary hypha close to the appressorium and the diameter at the apical region of the primary hypha before or after swelling. at all growth stages, the diameter of the primary hypha close to the appressorium was conserved with a mean of  μm. in infection sites with the anucleate primary hypha and a single nucleus present in the appressorium, the diameter of the primary hypha tip had a mean of  μm. in infection sites displaying one nucleus in the appressorium and another one in the bulbous portion of the primary hypha, the diameter of the bulbous portion had a mean of  μm, frequently ranging from to  μm. taken together, these data indicate that nuclear division and migration typically occur after the apical tip of the primary hypha has swollen to over 4 μm in diameter. mitosis in the appressorium and subsequent migration of a single nucleus into the first bulbous ih cell to determine nuclear dynamics during development of the first ih cell, we conducted time-lapse microscopy of rice cells invaded by m. oryzae mitotic reporter strain ckf expressing h1-tdtomato and gfp-nuclear localization signal. consistent with the previous study, after plant penetration, nuclear division occurred in the appressorium, and subsequently one mitotic nucleus migrated through the primary hypha to arrive in the swollen portion of the primary hypha, while the other nucleus remained in the appressorium. the timing of nuclear division was consistent with our prediction that nuclear division occurs in infections that had a swollen primary hypha tip lacking a nucleus. intriguingly, the nucleus that remained in the appressorium appeared to associate with the penetration pore as the other nucleus migrated through the primary hypha to the swollen hypha tip. our observation of nuclear division within the appressorium contrasts a previous report, in which the appressorial nucleus migrates to the primary hypha and then undergoes mitosis. we considered the possibility that the discrepancy might have arisen because different strains of m. oryzae and rice were used, specifically m. oryzae o-137-derived strain infecting rice strain yt in our study and m. oryzae guy11-derived strain infecting rice strain co- in the other study. however, we ruled out this possibility because we observed the consistent pattern of nucleus division within the appressorium followed by migration of a single nucleus into the primary hypha when m. oryzae guy11-derived strain infects yt or co-. the reason for this discrepancy remains to be determined. it is worth noting, however, that our findings are consistent with colletotrichum gloeosporioides, in which nuclear division occurs in the appressorium, and then one nucleus migrates into the penetration hypha, whereas the other nucleus remains in the appressorium.fig. nuclear division occurs within the appressorium. a m. oryzae ckf infecting rice cultivar yt shown are single plane confocal images of both red fluorescence and bright-field, and red fluorescence alone. nuclei are denoted by arrowheads. a single nucleus tagged with tdtomato separated into two mitotic nuclei within the appressorium. one nucleus then migrated out of focus into the primary hypha, while the other remained in the appressorium. b m. oryzae guy11-derived transformant infecting yt at the same growth stage as. nuclear dynamics were consistent with, with nuclear division occurring within the appressorium and migration of a single nucleus out of focus into the primary hypha. c m. oryzae guy11-derived transformant infecting rice cultivar co- at the same growth stage as both and. nuclear dynamics were again consistent with nuclear division occurring in the appressorium and migration of one nucleus into the primary hypha. bar = 5 μm formation of the first septum after nuclear division and migration to define the location and timing of septation during development of the first ih cell, we generated m. oryzae transformants expressing fluorescently labeled septa together with h1-tdtomato. the septa were visualized by constitutively expressing the plasma membrane-targeted plc delta:ph as a translational fusion to gfp in m. oryzae. plc delta:ph binds to pip lipids and has been shown to localize to the plasma membrane and septa in neurospora crassa. m. oryzae transformants expressing gfp-plc delta:ph showed green fluorescence localized at the plasma membrane of the appressorium and primary hypha. after nuclear division, one nucleus moved to the bulbous tip of the primary hypha, and the other nucleus remained in the appressorium. subsequently, the green fluorescence localized near the midpoint of the filamentous portion of the primary hypha, indicating the site of the first septum. the primary hypha was previously characterized by heath et al. and kankanala et al.: it is a filamentous structure that differentiates from the penetration peg, an extremely narrow structure that originates at the appressorium and forces through the plant cell wall, and that subsequently differentiates into bulbous ih. our septation result further reveals that the primary hypha is a filamentous part of two distinct cell types, constituting the invasive extension of the single-celled appressorium and the first ih cell.fig. the first post-penetration septum forms in the primary hypha. shown are maximum intensity projections of confocal optical sections of a rice cell invaded by m. oryzae strain ckf at hpi. merged nuclear tdtomato and gfp fused to plcdelta:ph are shown. top: one nucleus was located in the appressorium and gfp-plcdelta:ph was localized to the plasma membrane. middle: the same infection site imaged 50 min later showing one appressorial nucleus and one nucleus in the bulbous hypha. bottom: the same infection site after an additional 25 min showing gfp fluorescence concentrating at the location of the first septum. bar = 5 μm development of bics during hyphal differentiation two-stage development of bics has been previously documented: the tip bic is associated with the apex of the primary hypha; as the primary hypha switches to bulbous ih growth, the bic maintains its position on what is now the side of the bulbous ih. to determine how the tip bic becomes the side bic, we used our earlier observations about the time of nuclear division relative to hyphal swelling to design an experiment. we imaged m. oryzae transformant ckf expressing cytoplasmic eyfp and nuclear mrfp as well as mrfp fused to the bic-localized cytoplasmic effector pwl at different growth stages analogous to the three panels in fig. 1b. in cases of no bulbous differentiation, the bic was at the tip of the primary hypha. when bulbous differentiation had occurred, but nuclear division had not, the bic was already at the side, indicating that the bic is displaced from its tip position as the primary hypha swells. in infections where nuclear division and migration had taken place, we observed the bic still at the side, where it remained as a new hypha began to grow at the tip. these results indicate that the bulbous swelling of the primary hypha occurs prior to nuclear division and is responsible for the bic being “left” on the side of the cell. this displacement of the bic indicates that there is depolarization at the apex of the primary hypha during bulbous differentiation. the position of the bic on one side of the bic-associated cell suggests that the opposite side of the primary hypha grows more than the bic side. this asymmetric swelling produces an oval cell offset from the center of the hypha rather than a round cell, which would result from equal swelling in all directions. this growth is clearly distinct from the polarized growth observed during filamentous primary hypha development, of which the tip bic is characteristic. polarity in filamentous fungi is established and maintained by multiple proteins, especially those associated with control of cytoskeletal positioning. the spitzenkörper and polarisome have been established as instrumental in determining the direction of hyphal tip growth in filamentous fungi. in m. oryzae, spk-associated motor protein mlc is localized to the growing tip of the primary hypha before differentiation of the bic-associated cell. after differentiation, mlc is not present at the growing ih tip, but can be visualized near septa and in the subapical bic-associated cell. in contrast, the polarisome component spa is localized to the growing tip of the primary hypha and ih. because an ih cell develops from the growing end of the bic-associated cell, there must be a repolarization event after asymmetric swelling occurs. the spa and other polarisome proteins are likely involved in repolarization of the ih growing tip.fig. bic development. a tip to side bic differentiation and first nuclear division event at hpi. shown are maximum intensity projections of confocal optical sections. images are of m. oryzae ckf expressing cytoplasmic eyfp, nuclear mrfp, and bic-localized mrfp. arrows = bics. arrowheads = nuclei. asterisk = appressorium. left: tip bic stage. middle: side bic stage. right: after nuclear division, the side bic remained at the same position. bars = 5 μm. b representative measurement from the penetration point to the side bic in m. oryzae ckf at hpi. images show bic-localized mcherry, fungal hypha outlined by egfp, and mcherry and egfp overlapping in the side bic. top: a maximum intensity projection of confocal optical sections and 2-dimensional length measured with the open bezier tool in the zen black software. bottom: a 3-dimensional reconstruction of the same infection site shows 3d length measured with the imaris dendrite creation algorithm. bars = 5 μm. c comparative graphical summary of measurements taken with 3d imaris and with 2d zen black software of the distance between the penetration point to the side bic. individual infection sites have both a 3d measurement and a corresponding 2d measurement. the vertical distance between a data point and the 1-to- line on the graph corresponds to the difference in length between the 2d and 3d measurements associated with that data point regular growth from the appressorium penetration to the side bic the filamentous primary hypha appeared to extend from the appressorium to a consistent distance before widening at its apical region. we reasoned that the side bic would be positioned at a defined distance from the appressorium. to test this, we measured the distance from the appressorium penetration point to the side bic using m. oryzae transformant ckf expressing the bic-localized cytoplasmic effector pwl fused to mcherry:nls and also ih-outlining apoplastic effector bas fused to egfp. the penetration point was identified by examining the egfp fluorescence outlining the primary hypha in conjunction with the bright-field, and the bic was identified based on the strongly localized mcherry fluorescence. we conducted two-dimensional measurements of these images using the zen black open bezier tool. the distance along the primary hypha from the penetration point to the side bic has an average of 36 μm, ranging from to 45 μm. to obtain more accurate measurements of the same infection sites, we also used the three-dimensional dendrite measurement algorithm in imaris. an additional movie file shows the three-dimensional measurement in more detail. the average distance was 43 μm, ranging from to 54 μm. our finding of this considerable distance raises an intriguing question concerning effector secretion. cytoplasmic effectors such as pwl are secreted into the tip bic and the side bic, which are located more than 32 μm from the appressorial nucleus in which the pwl gene is expressed. there must be a mechanism to transport effectors from the appressorium through the primary hypha for their secretion to the distantly located bic, but the mechanism remains to be determined. additional file 2: movie s three-dimensional measurement from the appressorium penetration point to the bic of m. oryzae strain ckf growing in a rice cell at hpi. apoplastic effector bas with a translational fusion to egfp outlined the invasive hypha. the bic and the rice nucleus were visualized by bic-accumulating cytoplasmic effector pwl with a translational fusion to mcherry:nls. overlap of green and red signals is shown in yellow. source image is a series of 1 μm confocal optical sections taken over a depth of 23 μm. movie was created using the key frame animation feature in imaris software. at the start of the animation, the penetration point is on the left, and the bic is on the right. as the animation rotates, a penetration point-to-bic measurement is overlaid on the image. the measurement was performed with the dendrite creation algorithm in the imaris software and has a length of  μm. a maximum intensity projection of the same infection site in this movie is shown in fig. 4b, and a mirrored still of the last frame in this movie is shown in fig. 4b. bar = 5 μm. hyphal growth inside heat-killed rice cells we hypothesized that the development we observed during the early biotrophic invasion is specific to live host cells. to test this, we used a heat-killed cell assay, inoculating nonviable rice tissue with conidia. when inoculated on this tissue, m. oryzae produced the normal appressorium, indicating that the surface cues were still sufficient for appressorium differentiation. after penetration into heat-killed host cells, the fungus did not show distinct differentiation from filamentous to bulbous growth as observed during invasion of live host cells, but elongated, filamentous-like growth throughout the rice cell and its neighbors. hyphae growing in heat-killed cells spread rapidly into adjacent cells rather than completely filling the first cell, which the ih in living cells do over the course of 12 h. in living host cells, bics are easily recognized in bright-field images due to their predictable locations on the tip of the primary hypha, the side of the first bulbous ih cell, and the tips and sides of hyphae moving into adjacent host cells. we did not observe any indications of bics in the bright-field images of infections in heat-killed cells. the absence of bics in dead host cells is consistent with giraldo et al. showing that bics are derived from host membrane components such as the plasma membrane and endoplasmic reticulum; therefore, bics are unlikely to form in host cells, which lack intact membranes.fig. hyphal morphology in heat-killed rice cells is distinct from growth in live cells. m. oryzae ckf expressing cytoplasmic eyfp and nuclear tdtomato, growth in heat-killed rice cells at hpi. shown are merged fluorescence confocal images of a single focal plane, or a projection of the same infection site. rice cell walls are shown as white lines. top: a single focal plane shows the appressorium with a nucleus, germ tube, and hypha that grew on the rice surface. middle: a focal plane 12 μm beneath the appressorium shows filamentous-like ih branches and locations where ih had crossed the rice cell wall into neighboring cells. bottom: a maximum intensity projection shows all features of both focal planes. bar = 10 μm  we provide cellular details of early post-penetration development of m. oryzae during invasion of susceptible rice cells. this study defines developmental stages, in which the single-celled appressorium gives rise to the first ih cell, and the tip bic becomes the side bic when the tip bic-associated hyphal apex undergoes asymmetric swelling prior to mitosis in the appressorium. development of bics and bic-associated cells is induced during invasion of live rice cells. our study provides a descriptive framework for early biotrophic invasion of rice leaf sheath tissue. it remains to be determined whether the cellular processes reported in this study are conserved during infection of other rice tissues and of other plant species. this study also leads to new mechanistic questions concerning effector trafficking from the appressorium to the bic and hyphal differentiation in response to host environmental cues.fig. schematic diagram of early post-penetration growth in susceptible rice cells. pre-penetration: m. oryzae conidium germinates and forms an appressorium containing one nucleus. post-penetration: the appressorium penetrates the host cell wall, forming an anucleate filamentous primary hypha with the tip bic. the tip of the filamentous primary hypha swells and the tip bic becomes the side bic. the primary hypha remains anucleate. nuclear division occurs in the appressorium. one mitotic nucleus migrates into the bulbous portion of the primary hyphal tip, while the other nucleus remains in the appressorium. the first septum forms in the filamentous portion of the primary hypha, finalizing the establishment of the effector-secreting bic-associated cell. polarized growth resumes from the bic-associated cell 
to cause an economically important blast disease on rice, the filamentous fungus magnaporthe oryzae forms a specialized infection structure, called an appressorium, to penetrate host cells. our data raise new mechanistic questions concerning hyphal differentiation in response to host environmental cues and effector trafficking from the appressorium to the bic. the first ih cell, also known as the side bic-associated cell, is a specialized effector-secreting cell essential for a successful infection. the tip bic that was initially associated with the primary hypha became the side bic on the swollen apex prior to nuclear division in the appressorium. the average distance between the early side bic and the nearest nucleus in the appressorium was estimated to be more than 32 μm. these results suggest an unknown mechanism by which effectors that are expressed in the appressorium are transported through the primary hypha for their secretion into the distantly located bic. these hyphae secrete cytoplasmic effectors that enter host cells presumably via the biotrophic interfacial complex. appressoriumbiotrophic interfacial complexbiotrophyeffectorlive-cell imagingmitosisrice blast fungushyphal differentiationhttp://dx.doi.org/ /100005825national institute of food and agriculture2014-67013-21717khang chang hyun issue-copyright-statement© the author 2017 this study aims to determine cellular processes that lead to the development of this effector-secreting first ih cell inside susceptible rice cells. when m. oryzae was inoculated on heat-killed rice cells, penetration proceeded as normal, but there was no differentiation of a bulbous ih cell, suggesting its specialization for establishment of biotrophic infection. once inside host cells, the fungus produces a filamentous primary hypha that differentiates into multicellular bulbous invasive hyphae, which are surrounded by a host-derived membrane. the filamentous primary hypha extended from the appressorium and underwent asymmetric swelling at its apex. using live-cell confocal imaging, we determined a series of cellular events by which the appressorium gives rise to the first ih cell in live rice cells. septation occurred in the filamentous region of the primary hypha, establishing the first ih cell. our studies reveal cellular dynamics associated with the development of the effector-secreting first ih cell.
 to cause an economically important blast disease on rice, the filamentous fungus magnaporthe oryzae forms a specialized infection structure, called an appressorium, to penetrate host cells. once inside host cells, the fungus produces a filamentous primary hypha that differentiates into multicellular bulbous invasive hyphae, which are surrounded by a host-derived membrane. these hyphae secrete cytoplasmic effectors that enter host cells presumably via the biotrophic interfacial complex. the first ih cell, also known as the side bic-associated cell, is a specialized effector-secreting cell essential for a successful infection. this study aims to determine cellular processes that lead to the development of this effector-secreting first ih cell inside susceptible rice cells.  using live-cell confocal imaging, we determined a series of cellular events by which the appressorium gives rise to the first ih cell in live rice cells. the filamentous primary hypha extended from the appressorium and underwent asymmetric swelling at its apex. the single nucleus in the appressorium divided, and then one nucleus migrated into the swollen apex. septation occurred in the filamentous region of the primary hypha, establishing the first ih cell. the tip bic that was initially associated with the primary hypha became the side bic on the swollen apex prior to nuclear division in the appressorium. the average distance between the early side bic and the nearest nucleus in the appressorium was estimated to be more than 32 μm. these results suggest an unknown mechanism by which effectors that are expressed in the appressorium are transported through the primary hypha for their secretion into the distantly located bic. when m. oryzae was inoculated on heat-killed rice cells, penetration proceeded as normal, but there was no differentiation of a bulbous ih cell, suggesting its specialization for establishment of biotrophic infection.  our studies reveal cellular dynamics associated with the development of the effector-secreting first ih cell. our data raise new mechanistic questions concerning hyphal differentiation in response to host environmental cues and effector trafficking from the appressorium to the bic. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords appressoriumbiotrophic interfacial complexbiotrophyeffectorlive-cell imagingmitosisrice blast fungushyphal differentiationhttp://dx.doi.org/ /100005825national institute of food and agriculture2014-67013-21717khang chang hyun issue-copyright-statement© the author 2017 