Plant pathogenic fungi devised various strategies and mechanisms to infect plants. However, regardless of the pathogenic lifestyle, the initial stages of infection are common to most fungi: spores germinate on the host surface, undergo a short period of polarized growth, and then develop appressoria, which direct the growth into the plant tissues. Since the plant surface is poor in nutrients, it is assumed that fungi must utilize the spore's endogenous reserves to quickly move from the plant surface into the inner tissues. Various signals such as cutin monomers or surface physical features are known to induce appressoria formation, but it is unclear at which point post germination this switch from polar growth to swelling of the tip will occur. The short period of growth on the host surface is characterized by tight coupling of morphogenesis and cell cycle progression. On surfaces that support appressoria formation such as on plants or on a hydrophobic surface, appressoria will form on short germ tubes after a few nuclear divisions. On surfaces that do not support appressoria formation the germ tube will elongate and undergo many more nuclear and cell divisions. Thus, the number of cell divisions is not used by the fungus to determine the stage of appressorium formation without an appropriate surface signal. In this work we have studied the association between nuclei division and morphogenesis during pathogenic spore germination, appressorium formation, and appressorium-mediated plant penetration using a C. gloeosporioides transformant expressing Histone1-eGFP fusion protein.
Analysis of nuclear divisions revealed that mitosis was initiated immediately following exposure to germination-inducing signals. The first mitosis was observed 90 min after induction, which is similar to the elapsed time between the first and second mitoses. This suggests that in C. gloeosporioides, growth and cell division are inhibited in spores, but the spores are not dormant and can readily resume growth without a preceding adaptation stage upon receiving the proper signals. The growth inhibition is attributed to self-inhibitory compounds in the spore cell wall since removal of these substances by washing of the spores can enhance spore germination even without specific induction [1, 2]. The instant mitosis triggered during pathogenic germination differs from the pattern observed during saprophytic germination in C. gloeosporioides and other fungal species in which breaking of dormancy and isotropic swelling precedes mitosis and growth . In Candida albicans, cells that grow to high densities are arrested in G1. When diluted to lower densities the cells resume growth, but the first cell cycle is characterized by an extended G1 phase, which is about 70 min longer than the G1 phase in dividing cells . In Aspergillus nidulans, spores are dormant and germination is preceded by an early stage that lasts several hours and includes activation of metabolism and isotropic swelling [17, 22]. In C. gloeosporioides, saprophytic germination includes an initial period during which spores start swelling, but are neither growing nor dividing . In contrast, pathogenic germination has no adaptation stage; mitosis is tightly linked with germination response and seems to occur simultaneously or prior to cell growth.
We used a pharmacological approach in attempting to separate between the cell cycle and cell growth. Treatment of cells with HU always prevented the first nuclear division. This result indicates that in C. gloeosporioides the spores were arrested at G1, similar to the situation in other species [18, 23, 24]. The continuation of germination without mitosis is also in agreement with results from other species. In F. graminearum nuclear division is not required for spore germinating in rich medium . Under similar conditions, A. nidulans spore germination is tightly coordinated with nuclear division, but these two processes can be uncoupled under less favorable conditions . In Aspergillus fumigatus, cell cycle and morphogenesis are not interdependent, and it has been suggested that similar to yeast, these two processes run in parallel but share common checkpoints . Taken together, it seems that in most cases initiation of germination is independent of the cell cycle.
Blocking of mitosis in germinating spores of M. grisea using HU or by genetic intervention also lead to germination without nuclear division; however, appressoria formation was prevented . Similar results were reported in C. trancatum, where inhibition of the second round of DNA synthesis did not affect spore germination but blocked appressoria formation . In contrast, C. gloeosporioides spores continued to grow for at least 7–8 h in presence of HU, forming normal numbers of well-developed appressoria. Treatment of spores with benomyl also had no effect on germination rates, but growth was retarded at an earlier stage before appressoria formation. The early growth retardation following benomyl treatment is attributed to the role of microtubules in growth rather than to the lack of nuclei separation . These results suggest that in C. gloeosporioides all stages of pathogenic germination, including appressoria formation, are independent of mitosis, unlike M. grisea and C. trancatum in which appressoria formation and pathogenic development depend on completion of mitosis [14, 27]. Several other Colletotrichum species that we tested also produced high rates of appressoria in presence of HU, which were only slightly lower than the percentage of appressoria formation in untreated spores (Table 2). We noted that all of these species have small, unicellular spores whereas both M. grisea and C. trancatum have larger, three cells spores. At this point we do not know whether this structural difference has anything to do with the differences that we found in the regulation of cell cycle and appressoria formation.
We previously suggested a model in which plant signals bypass the effect of the self-inhibitory compounds and activate both mitosis and morphogenesis . Our current results suggest that induction of germination is unrelated to the induction of the cell cycle. This implies that either these two processes run in parallel and are simultaneously induced by the same signals, or that they might respond to different stimuli. In order to address these different possibilities, we used LatA to block cell growth and determined the effect on nuclear division. Unlike HU, which only blocked mitosis, LatA completely blocked both spore germination and nuclear division. When LatA was removed by washing of the spores, germination was resumed, showing that the effect was not a result of the toxicity of LatA but rather of the specific disruption of the actin cytoskeleton (data not shown). Thus, prevention of polar growth also leads to cell cycle arrest.
That morphogenetic development is necessary for continuation of the cell cycle is further supported by our observations that morphological changes always occurred before nuclear division. Following the first cell division, a germ tube emerged from one of the spore cells, and only then did the nucleus in this cell divide and migrate into the growing germ tube (Additional file 2). Similarly, the nucleus in the germ tube adjacent to the appressorium divided only when the appressorium was fully developed and it divided again only after the mature appressorium developed a penetrating hypha. Thus, in C. gloeosporioides morphogenesis always occurs before mitosis and the cell cycle depends on completion of the preceding morphogenetic stage.
According to these new results, we propose that plant signals specifically activate germination and that the cell cycle is induced only after initiation of germination. According to this model, the self-inhibitory compounds only inhibit growth, whereas cell cycle arrest is not directly affected by these compounds. This sequence of events is opposite from that in M. grisea, in which mitosis must be completed before appressorium formation, and the nucleus within the appressorium does not divide but migrates from the appressorium through the penetration peg into the primary hyphae . These differences are surprising, especially because M. grisea and C. gloeosporioides share a very similar infection strategy. Recently, Kankanala et al.  reported that M. grisea infects plant cells by moving biotrophic hyphae from one cell to another. This mode of infection differs from the infection mode of the true hemibiotroph Colletotrichum species, including C. gloeosporioides which use biotrophic hyphae to invade a few cells and then differentiate necrotrophic hyphae that kill the host cells . These differences, together with the difference in regulation of the early stages of infection, indicate that despite the apparent similarities in the sequence of events leading to pathogenesis, these fungi must have evolved very different regulatory mechanisms to control pathogenesis.
Another striking difference between C. gloeosporioides and M. grisea was our finding that the spores and appressoria of C. gloeosporioides remained viable throughout the infection cycle, unlike the spores of M. grisea, which were subjected to autophagy cell death soon after appressorium formation . This result is especially surprising since the unicellular spores of C. gloeosporioides are much smaller and therefore contain fewer resources than the three-celled spores of M. grisea. In several other fungal species it has been shown that spore components, and especially lipids and carbohydrates are recycled during germination . These works suggest that recycling of the spore components during germination is necessary for progression of the early stages of fungal development. In M. grisea mutants in the MAP kinase PMK1 and mitosis-defective mutants that are unable to form appressoria keep growing on the leaf surface without loosing viability . Additional M. grisea mutants also show this phenomenon, including G proteins and cAMP pathway mutants [31–33]. Moreover, in other fungi, mutants in similar genes were also able to develop considerable amounts of mycelia on the leaf surface [34, 35]. In C. gloeosporioides, spores that are germinated in rich medium do not develop appressoria and are not infective, but keep developing on the leaf surface . Collectively, these results show that when regulation of early pathogenesis is interrupted, fungal spores are capable of growing on the leaf surface for much longer periods of time than the very limited number of nuclear divisions, which normally precede appressoria formation. Therefore, the number of nuclear divisions prior to appressoria formation is not restricted by nutrient availability but rather, the limited growth of germ tubes before appressoria formation seems to constitute a common element in the regulation of early fungal pathogenesis. When the signal for growth arrest is missing, owing to mutations or lack of appropriate signals (e.g. on a hydrophilic surface or in rich medium), polar growth continues and mycelium develops. The endogenous nutrients within spores should be sufficient to support production of this biomass, or fungi must be able to absorb nutrients without plant penetration. Although we presently have no answers to these questions, we assume that both possibilities might occur. The final behavior of the spore, either for life or death, seems to be of variable importance; in some cases such as in M. grisea, it might be important, whereas in other cases such as in C. gloeosporioides, programmed cell death does not occur and is not required for plant infection.