BACKGROUND
infection of host plants by filamentous, appressoria forming fungi like the ascomycete colletotrichum higginsianum depends on directed, polarized growth and morphological switches of infection structures .
this fungus employs a hemibiotrophic infection strategy that includes two phases  <cit> .
after an initial biotrophic phase with bulbous primary hyphae, colletotrichum higginsianum switches to a necrotrophic growth phase forming characteristic thin secondary hyphae.
during penetration by the appressorium, which itself is a morphologically highly differentiated cell  <cit> , the tip of the penetration peg grows directed from the penetration pore towards the host epidermis  <cit> .
the infection vesicle and the primary hyphae grow out of this structure into the first host cell, which still has an intact plasma membrane and remains alive.
after the switch to necrotrophy, secondary hyphae develop.
these hyphae invade neighboring cells and grow strongly polarized at the hyphal tip similar to saprophytic hyphae of other filamentous fungi  <cit> .
for such processes, the establishment and maintenance of polarity and cell wall morphogenesis are critical.
in fungi, their regulation is influenced by pathways which include a central kinase and a mob-family protein that acts as co-activator  <cit> .
these pathways are called ram  and men  in s. cerevisiae  <cit>  or mor  and sin  in s. pombe  <cit> .
in s. cerevisiae, polarized growth, cell wall morphogenesis and other processes like cell separation, daughter cell-specific gene expression and cell cycle progression are influenced by the ram pathway  <cit> .
yeast cbk <dig> is the terminal kinase in this pathway and belongs to the nuclear dbf2-related /large tumor suppressor  kinase subfamily, which is conserved from yeast to man  <cit> .
mob <dig> binds to cbk <dig> and is essential for cbk <dig> kinase activity and its proper localization in yeast  <cit> .
until now, only some cbk <dig> targets have been identified.
e.g.
in yeast, cbk <dig> binds and phosphorylates sec <dig>  which is involved in polarized vesicle exocytosis  <cit> , and ssd <dig>  a mrna-binding protein that associates with many transcripts including chitinase cts <dig> mrna  <cit> .
aside from polarized cell morphology, a well-established function of the ram network in yeast is the control of activity and localization of the zinc finger transcription factor ace <dig> during late mitosis  <cit>  that regulates cell wall and cell separation genes like the chitinase cts <dig> and the glucanase scw <dig> specifically in the new daughter cell  <cit> .
nuclear localization of ace <dig> is also coordinated with the men signaling pathway  <cit> , which uses the ndr/lats kinase dbf <dig> and its associated kinase activator mob <dig>  <cit> .
the founding member of the ndr family, cot <dig>  is similar to yeast cbk <dig> and was identified in the filamentous fungus neurospora crassa as a temperature sensitive mutant allele  that showed impaired hyphal tip elongation  <cit> .
like its yeast homologs, cot <dig> requires binding of a mob co-activator protein for its activity.
this function appears to be mediated by two mob <dig> proteins in n. crassa, called mob2a and mob2b  <cit> .
filamentous fungi such as n. crassa and its close relative s. macrospora encode a third type of mob-family protein, called mob <dig>  <cit> .
mob <dig> is more similar to the mammalian striatin-binding protein phocein, does not seem to be involved in ndr signalling but was found to be important for the development of protoperithecia and for hyphal fusion .
previously, we described the identification of  <dig> c. higginsianum agrobacterium tumefaciens-mediated transformation  mutants, which are impaired in virulence on arabidopsis thaliana  <cit> .
here, we show that for one of these mutants , a hypomorphic allele of chmob <dig> is responsible for the virulence phenotype and we analyzed the function of the protein encoded by this locus in c. higginsianum.
we found that chmob <dig> is required for both conidiation and formation of functional appressoria and that it binds to the ndr/lats kinase chcbk <dig>  we searched for potential targets of the mob2/cbk <dig> complex and observed that mutants of two candidate genes have phenotypes similar to vir- <dig>  furthermore, we analyzed the roles of chmob <dig> and chmob <dig> in c. higginsianum.
RESULTS
vir- <dig> has defects in appressoria shape and production of conidia
vir- <dig> was isolated from a pool of t-dna insertion mutants with defects in virulence on arabidopsis thaliana  <cit> .
in contrast to arabidopsis infected with wild-type c. higginsianum where all leaves showed strong necrotic lesions after 4 days, infection with vir- <dig> resulted only in small and much fewer lesions .
staining infected leaves with trypan blue revealed that mutant vir- <dig> formed appressoria at levels comparable to wild-type, but they were often severely deformed .
at  <dig> dpi, nearly two thirds of the appressoria produced by vir- <dig> showed morphological defects like elongation, decreased diameter, small lateral protrusions or were less melanized.
some cells formed a melanized germ tube or a partly melanized conidium with or without an appressorium.
examples of these morphological phenotypes are shown in fig.
1b and additional file 1: figure s1a.
in contrast, the wild-type produced only 11% altered appressoria with less dramatic alterations .
the ability of morphologically abnormal appressoria to establish infectious primary hyphae was strongly reduced because only 10% of all primary hyphae emanated from abnormal appressoria with morphological defects .
as a consequence, the amount of primary hyphae formed by vir- <dig> at  <dig> dpi was reduced to only one third of the wild-type level .
the fraction of primary hyphae that gave rise to secondary hyphae was reduced by 62% in vir- <dig> .
on artificial hydrophobic plastic surfaces, appressoria of vir- <dig> were similarly deformed in 64% of the cases compared to 7% in the wt .fig.
<dig> t-dna insertion mutant vir- <dig> is impaired in virulence and conidiation.
a. thaliana col- <dig> plants were infected with c. higginsianum wt and the vir- <dig> mutant strain.
a macroscopic image at  <dig> dpi.
b microscopic images of germinated conidia with appressoria at  <dig> dpi on trypan blue stained leaves.
appressoria with unusual morphology are marked with white arrows.
c trypan blue stained a. thaliana leaves at  <dig> dpi.
d quantification of trypan blue stained primary hyphae  and secondary hyphae  at  <dig> dpi.
e appressoria of wt and vir- <dig> on artificial hydrophobic surface.
appressoria with unusual morphology are marked with arrows.
scale bars = 20 μm.
f vegetative growth of wt and vir- <dig> on oatmeal agar , potato dextrose agar  and czapek-dox minimal medium .
g, h average conidial cell volume g and conidial titer h of wt and vir- <dig> strains recovered from oatmeal agar after 7 days.
the data of  <dig> separate plates is given.
error bars are represented as the standard error from three replicates.
significant differences based on t-tests  are marked with asterisks
when inoculated on potato dextrose agar or minimal medium plates, vir- <dig> produced slightly smaller colonies than the wild-type .
furthermore, vir- <dig> did not show different sensitivity to several stress inducing compounds than the wild-type .
in addition to its virulence phenotype, mutant vir- <dig> generated less  conidia on oatmeal agar plates, which were significantly smaller than wild-type conidia .
taken together, the t-dna insertion mutant vir- <dig> is severely impaired in virulence due to defects during appressoria differentiation and host cell penetration.
vir- <dig> encodes a hypomorphic allele of chmob2
in order to identify the mutation responsible for the phenotype of vir- <dig>  we initially analyzed a t-dna insertion site found in the original mutant screen which suggested the presence of two linked t-dna insertions on supercontig <dig>  <cit>  close to ch063_ <dig> encoding a homolog to yeast mob <dig>  <cit> .
by pcr analyses using a series of primers specific to that locus, both regions flanking the insertion could be isolated and were sequenced .
the t-dna insertion site was located upstream of the atg in gene ch063_ <dig> and was accompanied by a small genomic deletion of 11 bp.
two t-dna copies were inserted in a head to head fashion .
attempts to amplify the junction between the two t-dna fragments by pcr were not successful, presumably due to the inverted arrangement of the sequences .
the presence of two t-dnas at this genomic context is consistent with the results of previous southern blot analysis probed with a hygromycin resistance dna fragment  <cit> .
ch063_ <dig> encodes a predicted  <dig> aa protein closely related to yeast mob <dig>  because the t-dna insertion was close to the annotated start codon and as the annotated n-terminus of ch063_ <dig> did not match the n-terminus of the yeast protein, it was important to verify the annotated gene model.
in order to identify the correct coding region and complete transcript, we performed 5’- and 3’-race pcr  using total wild-type rna as template .
as shown in fig.
2a, the resulting revised gene model for the chmob <dig> gene consists of two 49 nt introns in the coding region, a 5’-utr of 164 nt, a 3’-utr of 216 nt and 834 nt of coding sequence.
accordingly, the t-dna insertions of vir- <dig> are located 161 bp upstream of the chmob <dig> coding sequence.
interestingly, the putative chmob <dig> transcription start at - <dig>  as determined by 5’-race pcr, is located in the small genomic sequence which was replaced by t-dna sequence in the genome of vir- <dig>  since the insertion is positioned upstream of chmob <dig> and deletes the putative transcription start, we analyzed chmob <dig> mrna accumulation in rna samples from vir- <dig> and wild-type c. higginsianum by qualitative and quantitative pcr .
in wild-type samples, chmob <dig> rna was detected in conidia, in appressoria formed on artificial surfaces, in mycelium and in infected a. thaliana leaves.
rna levels reached 20% relative to the housekeeping gene chtubulin-α  in appressoria and 55% in rna from a. thaliana leaves 4 days after infection with wild-type c. higginsianum.
chmob <dig> was expressed strongly during appressoria formation as early as 4 h after inoculation .
in contrast, chmob <dig> transcript levels were reduced more than ten-fold in all vir- <dig> samples .
chmob <dig> expression in vir- <dig> infected a. thaliana could not be analyzed because virulence of this strain was too limited.fig.
<dig>
vir- <dig> encodes a hypomorphic chmob <dig> allele.
a schematic representation of the chmob <dig> locus in wild-type  and vir- <dig> .
beginning and end of the chmob <dig> transcript are shown relative to the start codon.
introns are depicted as striped boxes.
lb = left border; rb = right border.
b quantitative rt-pcr of chmob <dig> mrna abundance in conidia, appressoria, mycelium and in infected plants .
the data were normalized against α-tubulin  and are shown as the average of three biological replicates.
c semi-quantitative rt-pcr of chmob <dig> and α-tubulin in rna samples from in vitro appressoria  <dig>   <dig>   <dig> and 12 h after inoculation.
cdnas from wt, vir- <dig> and Δchace <dig>  strains were used as template for pcr.
genomic wt dna  or h2o were used as controls.
d, e
a. thaliana leaves at 3 days after infection with wt, vir- <dig> and vir- <dig> strains complemented with either the chmob <dig> wild-type allele  or with a ptef-mob2-gfp construct .
typical trypan blue stained leaves are shown in d. quantifications are shown in e. scale bars = 20 μm.
f average volume of conidia from the indicated strains recovered from oatmeal agar after 7 days.
the data from  <dig> separate plates is given.
error bars are represented as the standard error from three replicates.
significant differences based on t-tests  are marked with asterisks
in order to verify that the insertion upstream of chmob <dig> and the resulting strong reduction of chmob <dig> expression is responsible for the phenotype of the vir- <dig> mutant, we complemented vir- <dig> both with a plasmid  harboring the chmob <dig> wild-type allele from - <dig> to + <dig> and with a translational chmob2-gfp fusion under control of the strong and constitutive elongation factor  <dig> alpha promoter .
after reintroduction of wild-type chmob <dig> by atmt with pmob <dig>  virulence was fully restored .
when conidia were recovered from oatmeal plates, their cell titers  and average cell size were restored to nearly wild-type levels .
transformation with ptef1-mob2-gfp fully rescued the conidia cell size phenotype , while conidiation was restored to 80% of wild-type , similar to strains complemented with the wild-type allele.
in the ptef1-mob2-gfp transformants, virulence appeared to be even increased relative to the wild-type  possibly due to overexpression of chmob <dig>  in all cases, empty vector controls  did not affect the vir- <dig> phenotypes.
since all observed phenotypes of vir- <dig> can be complemented by reintroduction of chmob <dig>  the phenotype of this mutant must be linked to the identified insertion.
in summary, vir- <dig> encodes a hypomorphic chmob <dig> allele expressing less than 10% mrna, which was generated by insertion of t-dna in the 5’-utr separating the coding region from its promoter.
the c. higginsianum genome encodes three members of the mob1/phocein protein family
mob <dig> belongs to the mob1/phocein protein family, which is widely conserved among eukaryotes and many of its members have been shown to bind and activate ndr kinases  <cit> .
in contrast to metazoans, which contain up to six different mob proteins  <cit> , fungal genomes contain between two and four mob coding genes.
the resulting proteins can be grouped into three clades : the first contains proteins with high similarity to yeast mob <dig>  which binds the dbf <dig> kinase  <cit> , while the second clade clusters together with yeast mob <dig>  which binds to cbk <dig>  <cit> .
the third clade contains phocein, the founding member of the mob1/phocein family from rattus norvegicus  <cit> , and proteins from filamentous fungi like s. macrospora or n. crassa mob <dig>  which have been reported to be essential for development of reproductive protoperithecia and for hyphal fusion  <cit> .
all filamentous fungi analyzed in fig.
<dig> contain one protein from each clade, except s. macrospora and n. crassa, which both harbor two mob2-like genes.
in n. crassa, mob2a and mob2b have partly overlapping functions and were both shown to interact with the cbk <dig> like kinase cot <dig>  <cit> .
as c. higginsianum contains one protein from each clade, we assume that each mob protein has functions similar to the respective homologs from its clade.fig.
<dig> the genome of c. higginsianum encodes three mob-like proteins.
unrooted phylogenetic tree of mob proteins from c. higginsianum  and other fungi.
the tree was generated with the geneious tree builder plugin using a blosum <dig> cost matrix and the jukes-cantor tree building method.
the founding member of the mob family  from rat was included as a reference.
branch lengths correspond to substitutions per site.
scale bar =  <dig>  substitutions per site
chmob <dig> and chcbk <dig> may be essential genes in c. higginsianum
in order to obtain Δchmob <dig> null mutants, we attempted to perform targeted gene knockout of chmob <dig> by transformation of the non-homologous end-joining defective mutant Δchku <dig>  <cit>  with the chmob <dig> deletion plasmid pck <dig>  this plasmid harbors the hygromycin resistance cassette  flanked by chmob <dig> upstream  and downstream sequence  on the t-dna.
in the Δchku <dig> background >90% of all transforming dna should integrate by homologous recombination  <cit> .
in three independent experiments, however, we recovered only transformants that harbored ectopically integrated dna .
in contrast, transformations of the nhej proficient wild-type strain yielded > <dig> transformants.
we therefore expect that chmob <dig> may be an essential gene in c. higginsianum, at least in the Δchku <dig> background.
interestingly, mob <dig> can be inactivated in s. cerevisiae and in n. crassa  <cit> .
as described for s. cerevisiae, mob family proteins often act as co-activators for ndr/lats kinases.
in particular, mob <dig> binds to cbk <dig> kinase in yeast  <cit> .
we assumed that chmob <dig> has a similar function in c. higginsianum and may act through activation of chcbk <dig>  we attempted to delete chcbk <dig>  in the Δchku <dig> background.
in three independent experiments this failed similar to chmob <dig> .
it is therefore likely that both chmob <dig> and chcbk <dig> have essential functions in c. higginsianum.
in addition, attempts to generate knock-down mutants of these genes by expression of antisense-rnas did not lead to transformants with significantly reduced levels of the respective transcripts .
chmob <dig> co-localizes with the potential kinase chcbk <dig> in appressoria
in order to further examine the function of c. higginsianum mob <dig>  we investigated the subcellular localization of chmob <dig> and its potential binding partner chcbk <dig>  we constructed a strain  where the genomic copy of chcbk <dig> was replaced by a cbk1-mcherry fusion and where chmob <dig> was simultaneously replaced by mob2-gfp.
the resulting strain had no significant defects in either virulence, conidiation or vegetative growth except for producing slightly smaller conidia .
since both chmob <dig> and chcbk <dig> may be essential in c. higginsianum, reduced chmob <dig> or chcbk <dig> function should have obvious effects.
we therefore concluded that both fusion proteins were functional.
in conidia isolated from oma plates , both fusion proteins were more or less evenly distributed over the whole cell except for the nucleus, which appeared to be partly excluded.
the two proteins co-localized to some extent.
early upon in vitro appressoria formation, when appressoria were unmelanized , both cbk1-mcherry and mob2-gfp were completely overlapping.
in the germinated conidia attached to these appressoria, however, the fusion proteins seemed to be localized to different compartments.
in mature appressoria and their corresponding germinated conidia , both fusion proteins were again co-localized all over the cells except in nuclei, where both were absent.
in axenic cultures, co-localization could not be addressed because the cbk1-mcherry fusion was barely detectable .
in planta, we could not detect either of the two fusion proteins, presumably because of the high background fluorescence in green leaves .
taken together, mob2-gfp and cbk1-mcherry show the same subcellular localization during appressoria formation.fig.
<dig> chmob <dig> co-localizes with chcbk <dig> in the cytoplasm.
conidia of Δchcbk1:: cbk1-mcherry Δchmob2:: mob2-gfp  were rinsed off oatmeal plates and analyzed by clsm during in vitro appressoria formation.
a conidia, b in vitro ap at  <dig> hpi, c in vitro ap at  <dig> hpi.
bright field, gfp and mcherry channels are shown from left to right.
the rightmost picture shows an overlay of gfp and mcherry channels.
the intensities between panels are not directly comparable, because different settings were used to optimally visualize protein localization.
scale bars = 20 μm
mob2-gfp physically interacts with cbk1-ha
chmob <dig> and chcbk <dig> can occupy the same subcellular compartment.
consequently, they might physically interact with each other during certain times in the cell cycle.
in order to test their possible interaction, we performed co-immunoprecipitation of chmob <dig> and chcbk <dig>  we constructed a strain  where chcbk <dig> was fused to a 6xha tag at its own genetic locus.
in addition, this strain ectopically expressed mob2-gfp under control of the tef1a promoter.
both proteins could be detected in axenic mycelium after western blotting with anti-gfp and anti-ha antibodies .
mob2-gfp was detected at about 60 kda .
cbk1-ha produced several bands between  <dig> and 95 kda, possibly because of different phosphorylation states of the protein or due to protein degradation .
we used protein extracts from mycelium to investigate the interaction of chcbk <dig> with chmob <dig>  first, cbk1-ha was immunoprecipitated with anti-ha antibodies coupled to magnetic beads .
the successful pull down of cbk1-ha is shown in the western blot decorated with anti-ha antibodies .
the presence of mob2-gfp in these immunoprecipitates was determined by decorating the western blots with anti-gfp antibodies .
in extracts containing both fusion proteins , mob2-gfp was efficiently co-immunoprecipitated with cbk1-ha , but not from control extracts lacking cbk1-ha .
similarly, when mob2-gfp was pulled down with anti-gfp antibody coated beads, cbk1-ha was co-precipitated .
some cbk1-ha bands were missing from the anti-gfp ip, suggesting that at least some of the chcbk <dig> forms might not bind to chmob <dig>  the successful co-precipitation of chmob <dig> and chcbk <dig> indicates that the two proteins can interact in axenic c. higginsianum cells.
while technically not testable, it is likely that this interaction also occurs during infection.fig.
<dig> chmob <dig> interacts with chcbk <dig>  whole cell extracts from strains expressing different combinations of chmob2-gfp and chcbk1-ha were used to analyze the interaction of chmob <dig> and chcbk <dig> by immunoprecipitation.
strains used from left to right: Δchcbk1:: cbk1-ha ptef1a-mob2-gfp , the parental strain Δchku <dig> , Δchcbk1:: cbk1-ha  and ptef1a-mob2-gfp .
western blots of whole cell protein extracts , immunoprecipitates with anti-ha antibody coated beads  and immunoprecipitates with anti-gfp antibody coated beads  were decorated either with anti-gfp  or with anti-ha  primary antibodies.
the band corresponding to mob2-gfp is marked with an arrow.
the multiple bands corresponding to cbk1-ha are marked with a bracket.
asterisks mark bands corresponding to primary antibody which are present in the ip and therefore are recognized by the secondary antibody
next, we tried to verify this interaction by mass spectrometry using cbk1-ha expressing cell extracts without overexpressing mob2-gfp.
this approach could potentially also identify downstream targets of chcbk1-phosphorylation and could show whether or not chmob <dig> is the sole activator of chcbk <dig>  we pulled down cbk1-ha from extracts of axenic mycelium of cy <dig>  and from extracts of the parental strain as control in two biological replicates using anti-ha antibody-coated magnetic beads.
the eluates were analyzed by nano lc-ms/ms and quantified label-free by integrating peak areas .
chmob <dig> was only identified in the cbk1-ha ips but not in samples from the untagged parental strain, further verifying the interaction of these two proteins in c. higginsianum.
the other peptides identified were probably contaminants as they consisted mainly of highly expressed enzymes from the citrate cycle, from glycolysis or proteins associated with ribosomes.
the lack of potential chcbk <dig> targets in the pull-downs is likely due to transient interactions of the kinase with its substrates.
alternatively, the important proteins may not be present or not targeted by chcbk <dig> in samples of in vitro mycelium.
two potential downstream targets of the chmob2/chcbk <dig> complex are required for full pathogenicity
two well described cbk <dig> targets in yeast are the transcription factor ace <dig> and the mrna-binding protein ssd <dig>  <cit> .
we therefore tested whether or not their orthologs from c. higginsianum are involved in pathogenicity.
we identified possible orthologs in the c. higginsianum genome  <cit> .
the closest homolog to scace <dig> was ch063_ <dig> with  <dig> % identity.
after comparison with the sequence of s. cerevisiae ace <dig> and the more closely related aface <dig> of a. fumigatus  <cit> , we concluded that the automatic annotation of ch063_ <dig> was not complete and extended the n-terminus by  <dig> amino acids resulting in a revised c. higginsianum chace <dig> gene model encoding for  <dig> amino acids.
this extension could be verified by sequencing chace <dig> cdna .
the revised protein has  <dig> % sequence identity to scace <dig> and  <dig> % to aface <dig> over the full sequence.
the region containing the c2h <dig> zinc finger dna binding domain showed  <dig> % sequence identity to aface <dig>  the potential c. higginsianum ssd <dig>  shows  <dig> %,  <dig> % and  <dig> % sequence identity to the potential orthologs from saccharomyces cerevisiae , magnaporthe grisea  and colletotrichum lagenarium , respectively.
although both chssd <dig> and chace <dig> show less than 40% identity to their respective yeast homologs, they are clearly the best match in reciprocal blast searches .
we generated Δchace <dig>  and Δchssd <dig>  deletion strains in the Δchku <dig> background and examined their phenotype .
consistent with a role of chace <dig> and chssd <dig> downstream of the chmob2/chcbk <dig> complex, both mutants exhibited significantly reduced virulence .
furthermore, Δchace <dig> and Δchssd <dig> strains showed decreased vegetative fitness, as their colonies on minimal medium were smaller by about 35% and 50% than wild-type, respectively .
none of the mutants showed significantly altered sensitivity to osmotic stress, heat, h2o <dig>  or cell wall stress .
interestingly, conidial cell size and especially the conidial titer recovered from oma plates were reduced in the Δchace <dig> mutant  similar to the phenotype observed in vir- <dig>  the phenotype of Δchace <dig> could be rescued by reintroduction of wild-type chace <dig> into the Δchku <dig> locus of this strain , further confirming the involvement of chace <dig> in virulence and conidiation.
the decrease in fitness of the Δchace <dig> and Δchssd <dig> mutants could contribute to their virulence phenotype.
the conidiation phenotype of the Δchace <dig> mutant, however, is likely to be a specific phenotype, as slow growing mutants do not a priori produce less conidia .
it was not possible to generate a Δchace <dig> Δchssd <dig> double mutant .fig.
<dig>
chace <dig> and chssd <dig> are required for full virulence of c. higginsianum.
a schematic representation of the genomic loci of chace <dig> and chssd <dig>  and the knockout cassettes of the respective deletion plasmids .
the position of 5’ and 3’ flanking regions are given relative to the respective start codon.
b spray infection of a. thaliana at  <dig> dpi.
primary hyphae  and secondary hyphae  produced by the indicated strains.
c vegetative growth of the indicated strains on potato dextrose agar  and czapek-dox agar minimal medium .
d average number of conidia  and cell volume  for the indicated strains recovered from oatmeal agar after 7 days.
the data from  <dig> separate plates is shown.
the strains used in b-d were wild-type , vir- <dig>  Δchace <dig> , Δchssd <dig>  and the Δchku <dig> parental strain of the last two .
error bars are represented as the standard error from three replicates.
significant differences based on t-tests  are marked with asterisks
in order to analyze if chace <dig> is regulated by the chmob2/chcbk <dig> complex in c. higginsianum as in s. cerevisiae  <cit> , we analyzed the mrna amount of potential orthologs of the scace <dig> targets cts <dig> and scw <dig>  <cit>  and of the s. pombe ace <dig> target mid <dig>  <cit>  in samples from vir- <dig> and Δchace <dig>  we expected that if chace <dig> activity were regulated by chmob <dig>  the transcript amount of chace <dig> targets might be different in vir- <dig>  which expresses only little chmob <dig> , and in Δchace <dig> mutants, which lacks chace <dig>  however, using both semi-quantitative and quantitative rt-pcr we could not detect significant chcts <dig>  chscw <dig> or chmid <dig> transcript changes in appressoria samples in comparison to the wild-type .
the mrna level of chace <dig> was not significantly different between vir- <dig> and the wild-type .
this could indicate that similar to the situation in yeast  <cit> , chace <dig> is not regulated on the level of mrna accumulation but by chcbk1-dependent phosphorylation.
furthermore, Δchcts <dig> strains , which lack one of the genes predicted to encode chitinase, did not show any defects in virulence, conidiation or cell separation .
a cell separation phenotype was observed in yeast Δcts <dig> mutants  <cit> .
this may indicate that ace <dig> regulates different targets in c. higginsianum than it does in yeasts.
in summary, we were not able to identify downstream targets of chmob2/chcbk <dig> in c. higginsianum, but deletion of two cbk <dig> targets known from other systems resulted in pathogenicity and conidiation phenotypes resembling those of vir- <dig>
the roles of chmob <dig> and chmob <dig> in c. higginsianum
while mob <dig> and mob <dig> seem to be present in all fungal genomes, mob <dig> is only found in filamentous fungi and higher eukaryotes  <cit> .
the mammalian ortholog of mob <dig>  was identified in rat dendritic cells and has been shown to bind to striatin, a calmodulin binding protein  <cit> .
in fungi, mob <dig> has been reported to be essential for sexual development and for hyphal fusion in n. crassa and s. macrospora  <cit> .
although c. higginsianum has not been described to perform a sexual cycle, a potential ortholog of this gene is encoded in the c. higginsianum genome.
we therefore investigated the potential roles of chmob <dig> in c. higginsianum by targeted gene knockout of chmob <dig> .
except for a very mild reduction in colony size on both pda and minimal medium, we did not observe significant changes either in virulence or conidiation of the resulting Δchmob <dig> mutants .
the expression pattern of chmob <dig> did not give any further hint regarding its physiological function, as it was expressed at low, constitutive levels under all tested conditions .
we cannot exclude that chmob <dig> has a specific function under different, specific conditions.
in yeasts and n. crassa, mob <dig> and mob <dig> have distinct, but similar functions, namely the binding and activation of a ndr/lats-kinase  <cit> .
in s. cerevisiae, mob <dig> binds to cbk <dig> in the ram pathway and mob <dig> binds to dbf <dig> in the mitotic exit network.
although binding of mob <dig> or mob <dig> to their respective kinases is thought to be specific  <cit> , there may be crosstalk between men and ram networks in s. cerevisiae  <cit> .
we therefore investigated if chmob <dig> and chmob <dig> have overlapping functions in c. higginsianum by genetically inactivating chmob <dig> .
transformation of Δchku <dig> with a chmob <dig> deletion plasmid resulted in mutants with a strong growth phenotype on all tested media .
in addition, these mutants showed strong sensitivity to the cell wall stress inducing and chitin binding dye congo red , indicating a defect in chitin turnover in the cell wall  <cit> .
furthermore, Δchmob <dig> mutants hardly produced any conidia on oatmeal medium .
the few conidia that could be recovered were smaller by about 50%  and were significantly reduced in formation of appressoria, primary hyphae and secondary hyphae after droplet inoculations .
in contrast to wild-type conidia which form a septum during germination, Δchmob <dig> conidia were never septated .
furthermore, Δchmob <dig> conidia were often connected to  <dig> or  <dig> other conidia, possibly because they did not undergo proper cell separation during conidiation .
hyphae from axenic mycelium of Δchmob <dig> strains also hardly produced any septae at all, in contrast to the parental strain .
the strong septation phenotype of Δchmob <dig> strains is similar to mutants in s. cerevisiae and s. pombe, where mob <dig> is required for completion of mitosis  <cit> , initiation of cytokinesis and septation  <cit> .fig  <dig>
chmob <dig> is required for septum formation and cell division.
two independent Δchmob <dig> transformants  and the parental strain Δchku <dig>  were tested for vegetative growth, virulence and the ability to produce septae.
a vegetative growth on oatmeal agar , potato dextrose agar  and czapek-dox minimal medium .
b
a. thaliana 4 days after droplet inoculation.
representative images of trypan blue stained leaves are shown on the right.
the quantification of appressoria , primary hyphae  and secondary hyphae  of this infection is shown on the left.
the data of  <dig> plants per strain with  <dig> leaves per plant with at least  <dig> counted appressoria per leaf is given.
error bars are represented as the standard error from three replicates.
significant differences based on t-tests  are marked with asterisks.
c axenic mycelium stained with calcofluor white.
septae are marked with white arrows.
scale bars = 20 μm
in summary, c. higginsianum contains two additional mob genes.
consistent with a function of mob <dig> during sexual development in other systems  <cit> , deletion of chmob <dig> did not lead to any significant phenotype in c. higginsianum which is lacking a sexual cycle.
in contrast, knockout of chmob <dig> leads strong defects in appressoria formation, vegetative growth, septation, conidiation and infection.
discussion
screening for genes involved in pathogenicity though aimed at identification of genes with functions exclusively required for certain aspects of the infection process, will always also identify genes with vegetative functions at many stages of their life cycle.
atpases  <cit> , intracellular transport  <cit> , nutrition  <cit>  and morphogenesis  <cit>  are notable examples.
appressorial pathogens like colletotrichum higginsianum undergo switches in polarity during infection of plant cells.
in particular, the directed formation of a penetration peg underneath the appressorium is essential for infection.
it therefore is not too surprising to identify polarity determinants like mob <dig> in a screen for pathogenicity genes.
we identified a weak allele of chmob <dig> in the vir- <dig> mutant that in most cases produces appressoria with morphologic defects like elongation or secondary outgrowths.
these appressoria are not able to penetrate and to initiate the infection process.
since a fraction of the vir- <dig> appressoria looked normal and appeared functional, the mutant shows residual weak symptoms in some spots on the leaf, resulting in a mutant with reduced pathogenicity.
in addition, vir- <dig> produces less and smaller conidia.
although vir- <dig> expresses less than 10% of chmob <dig> mrna relative to the wild-type, it is not a null mutant.
since the t-dna insertion in the genome of vir- <dig> is located just in front of the chmob <dig> reading frame, it is likely that residual chmob <dig> activity may be present, at least in some cells.
the phenotype of a true null mutant could not be analyzed because all attempts to generate a deletion allele by homologous recombination failed.
the likely explanation, therefore, is that chmob <dig> is an essential gene at least in the Δchku <dig> background used for all knockouts.
a low expression, which may vary from cell to cell, could also be the reason why not all mutant cells behave identical, resulting in incomplete penetrance of the underlying genetic defect.
while this makes interpretation of the phenotype more difficult, the fortuitous isolation of a weak allele allowed us to characterize the functions of chmob <dig>  the defect of most vir- <dig> appressoria to penetrate the host tissue correlated with morphological defects.
in agreement with a possible essential function for chmob <dig>  its binding partner, the ndr/lats kinase chcbk <dig>  could also not be knocked out.
while a failure to generate knockout mutants is no evidence for an essential function, it resembles the situation in yeast and s. pombe, where mob <dig> and cbk <dig> have been found to be essential.
<cit> .
interestingly, the lethality of ram mutations in yeast is suppressed by loss of ssd <dig> , showing that the consequences of ram mutations may be strain specific.
in most systems, mob <dig> and cbk <dig> mutations are indeed associated with pleiotropic phenotypes and morphological defects.
in the opportunistic human pathogen c. albicans, all mutants in the ram pathway including camob <dig> and cacbk <dig> are viable but show hyperpolarization  <cit> .
in the basidiomycetes c. neoformans and u. maydis, mutants of the cbk <dig> ortholog ukc <dig>  mob <dig> and all other associated proteins thought to be involved in the same pathway lead to hyperpolarized growth and decreased virulence  <cit> .
in a. nidulans, mutants in cbk <dig>  and mob <dig> orthologs  show growth defects, strongly reduced ability to form conidia, altered conidia cell size and increased number of nuclei in spores  <cit> .
in n. crassa, which is more closely related to c. higginsianum, the cot- <dig>  mutant and the mob2a mob2b double mutant show increased rate of branching in axenic mycelium, reduced growth rate and reduced conidiation  <cit> .
most phenotypes observed in our study for the chmob <dig> mutant vir- <dig> are consistent with functions reported for mob <dig> and cbk <dig> in filamentous fungi, including a function in conidiation.
reduced cell size of conidia was an obvious phenotype of vir- <dig>  altered conidial cell size may be the consequence of altered cell division or septation in conidial precursor cells  <cit> .
altered conidial cell size was also observed in n. crassa cot <dig> mutants.
they were, however,  <dig> times larger than wild-type cells, rather than smaller as in the case of c. higginsianum  <cit> .
we also observed about 50% reduction in conidiation ability in the vir- <dig> mutant.
considering that vir- <dig> is not a null allele of chmob <dig>  this phenotype is reminiscent of a. nidulans and n. crassa mob <dig> mutants which largely fail to form conidia  <cit> .
conidiation is critically dependent on septation, which in turn is also dependent on the mob1/dbf2-dependent men  or sin  signalling pathways in many fungi .
in fact, n. crassa and a. nidulans
Δmob <dig>  <cit>  show sporulation defects similar to the c. higginsianum Δchmob <dig> mutants described in this study.
it is possible that the mob1/dbf <dig> and mob2/cbk <dig> complexes have overlapping functions or that they control each other.
the latter may be the case in related systems.
in n. crassa, mob <dig> appears to affect cot <dig> activity more than its direct interactor and co-activator mob2b  <cit> .
in s. pombe, spsid <dig>  controls sporb <dig>  activity  <cit> , while mob <dig> and cdc <dig> activity is required for proper mob <dig> and ace <dig> localization in yeast  <cit> .
for comparison, a simplified model of the potential interplay between men and ram of s. cerevisiae is shown in additional file 10: figure s <dig>
in addition to a strong conidiation phenotype, Δchmob <dig> mutants exhibited attenuated ability to form appressoria, primary and secondary hyphae upon infection and showed a severe septation defect.
these phenotypes are similar to the corresponding mutants in n. crassa  <cit>  and a. nidulans  <cit> .
mob <dig> is essential both in s. cerevisiae  <cit>  and s. pombe  <cit> , but cells harboring conditional alleles of mob <dig> either arrest in late mitosis or, in the case of s. pombe, do not septate and become multinucleate.
in addition to nuclear division, s. cerevisiae mob <dig> also seems to have a role during cytokinesis and cell separation  <cit> .
based on these similarities, it is likely that the function of mob <dig> and its associated signalling pathway is conserved among ascomycetes.
what are the critical targets of the mob2/cbk <dig> pathway in filamentous fungi?
similar to the situation in yeast, it was found that deletion of the n. crassa homolog of ssd <dig>  can suppress defects of the cot <dig>  allele  <cit>  suggesting that ssd <dig> is an important component of the ram pathway also in filamentous fungi.
in s. cerevisiae, ssd <dig> inactivation is thought to occur through cbk <dig> phosphorylation .
because ssd <dig> may be a conserved cbk <dig> target we analyzed the phenotype of a Δchssd <dig> mutant allele.
we found that the lack of chssd <dig> leads to attenuated virulence in c. higginsianum.
the effect on virulence was similar to vir- <dig> and to Δssd <dig> mutants in the plant pathogens c. lagenarium and magnaporthe grisea  <cit>  supporting the notion that ssd <dig> is a conserved virulence associated target of the ram pathway.
in c. lagenarium  <cit> , Δssd <dig> mutants mostly failed to penetrate and intracellular hyphae were only detected very rarely.
in m. grisea  <cit> , Δssd <dig> mutants were still able to produce intracellular primary hyphae on rice plants, but they were mostly restricted to dead host cells.
we also analyzed the effect of chace <dig> on pathogenicity in c. higginsianum because it may be a conserved target of the ram pathway.
again, we saw a reduction of pathogenicity and, interestingly, a comparable effect on cell volume in conidia and reduced conidiation.
however, we found no evidence for posttranslational regulation of chace <dig> by the chmob2/chcbk <dig> complex, as described for s. cerevisiae  <cit> .
we observed that c. higginsianum mob <dig> and cbk <dig> localize to the cytoplasm and are excluded from nuclei in conidia and during in vitro appressoria formation.
in baker’s yeast, however, mob <dig> and cbk <dig> proteins can be found at sites of polarized growth like the cortex of the growing bud and the mating projection or in the daughter cell nucleus and the septum during cell separation, where they supposedly regulate cytokinesis genes through the transcription factor scace <dig>  <cit> .
this difference in localization could indicate that ace <dig> is not a conserved mob2/cbk <dig> target in filamentous fungi.
in the absence of verified targets for the transcription factor chace <dig>  it also remains unclear whether or not chace <dig> and yeast ace <dig> have a common function.
a protein homologous to scace <dig> was also analyzed in a. fumigatus  <cit> , where knockout mutants had attenuated ability to produce conidia, abnormal cell wall architecture and were hypervirulent in a mouse model.
this protein  is more closely related to chace <dig>  than to the yeast protein and, given their common conidiation phenotype, they may be orthologous gene products.
CONCLUSIONS
this study showed that the mob-family protein chmob <dig> from the plant pathogen colletotrichum higginsianum is involved in virulence on arabidopsis thaliana and has a role in conidiation.
chmob <dig> forms a complex with the conserved chcbk <dig> kinase.
the study further analyzed the functions of the other two mob proteins encoded in c. higginsianum by targeted gene knockouts.
chmob <dig> is required for conidiation, cytokinesis and plant infection, while Δchmob <dig> mutants have no obvious phenotype in vegetative cells or during infection.

 infection of host plants by filamentous, appressoria forming fungi like the ascomycete colletotrichum higginsianum depends on directed, polarized growth and morphological switches of infection structures. this fungus employs a hemibiotrophic infection strategy that includes two phases. after an initial biotrophic phase with bulbous primary hyphae, colletotrichum higginsianum switches to a necrotrophic growth phase forming characteristic thin secondary hyphae. during penetration by the appressorium, which itself is a morphologically highly differentiated cell, the tip of the penetration peg grows directed from the penetration pore towards the host epidermis. the infection vesicle and the primary hyphae grow out of this structure into the first host cell, which still has an intact plasma membrane and remains alive. after the switch to necrotrophy, secondary hyphae develop. these hyphae invade neighboring cells and grow strongly polarized at the hyphal tip similar to saprophytic hyphae of other filamentous fungi. for such processes, the establishment and maintenance of polarity and cell wall morphogenesis are critical. in fungi, their regulation is influenced by pathways which include a central kinase and a mob-family protein that acts as co-activator. these pathways are called ram and men in s. cerevisiae or mor and sin in s. pombe. in s. cerevisiae, polarized growth, cell wall morphogenesis and other processes like cell separation, daughter cell-specific gene expression and cell cycle progression are influenced by the ram pathway. yeast cbk is the terminal kinase in this pathway and belongs to the nuclear dbf2-related /large tumor suppressor kinase subfamily, which is conserved from yeast to man. mob binds to cbk and is essential for cbk kinase activity and its proper localization in yeast. until now, only some cbk targets have been identified. e.g. in yeast, cbk binds and phosphorylates sec which is involved in polarized vesicle exocytosis, and ssd a mrna-binding protein that associates with many transcripts including chitinase cts mrna. aside from polarized cell morphology, a well-established function of the ram network in yeast is the control of activity and localization of the zinc finger transcription factor ace during late mitosis that regulates cell wall and cell separation genes like the chitinase cts and the glucanase scw specifically in the new daughter cell. nuclear localization of ace is also coordinated with the men signaling pathway, which uses the ndr/lats kinase dbf and its associated kinase activator mob. the founding member of the ndr family, cot is similar to yeast cbk and was identified in the filamentous fungus neurospora crassa as a temperature sensitive mutant allele that showed impaired hyphal tip elongation. like its yeast homologs, cot requires binding of a mob co-activator protein for its activity. this function appears to be mediated by two mob proteins in n. crassa, called mob2a and mob2b. filamentous fungi such as n. crassa and its close relative s. macrospora encode a third type of mob-family protein, called mob. mob is more similar to the mammalian striatin-binding protein phocein, does not seem to be involved in ndr signalling but was found to be important for the development of protoperithecia and for hyphal fusion. previously, we described the identification of c. higginsianum agrobacterium tumefaciens-mediated transformation mutants, which are impaired in virulence on arabidopsis thaliana. here, we show that for one of these mutants, a hypomorphic allele of chmob is responsible for the virulence phenotype and we analyzed the function of the protein encoded by this locus in c. higginsianum. we found that chmob is required for both conidiation and formation of functional appressoria and that it binds to the ndr/lats kinase chcbk we searched for potential targets of the mob2/cbk complex and observed that mutants of two candidate genes have phenotypes similar to vir- furthermore, we analyzed the roles of chmob and chmob in c. higginsianum.  vir- has defects in appressoria shape and production of conidia vir- was isolated from a pool of t-dna insertion mutants with defects in virulence on arabidopsis thaliana. in contrast to arabidopsis infected with wild-type c. higginsianum where all leaves showed strong necrotic lesions after 4 days, infection with vir- resulted only in small and much fewer lesions. staining infected leaves with trypan blue revealed that mutant vir- formed appressoria at levels comparable to wild-type, but they were often severely deformed. at dpi, nearly two thirds of the appressoria produced by vir- showed morphological defects like elongation, decreased diameter, small lateral protrusions or were less melanized. some cells formed a melanized germ tube or a partly melanized conidium with or without an appressorium. examples of these morphological phenotypes are shown in fig. 1b and additional file 1: figure s1a. in contrast, the wild-type produced only 11% altered appressoria with less dramatic alterations. the ability of morphologically abnormal appressoria to establish infectious primary hyphae was strongly reduced because only 10% of all primary hyphae emanated from abnormal appressoria with morphological defects. as a consequence, the amount of primary hyphae formed by vir- at dpi was reduced to only one third of the wild-type level. the fraction of primary hyphae that gave rise to secondary hyphae was reduced by 62% in vir-. on artificial hydrophobic plastic surfaces, appressoria of vir- were similarly deformed in 64% of the cases compared to 7% in the wt.fig. t-dna insertion mutant vir- is impaired in virulence and conidiation. a. thaliana col- plants were infected with c. higginsianum wt and the vir- mutant strain. a macroscopic image at dpi. b microscopic images of germinated conidia with appressoria at dpi on trypan blue stained leaves. appressoria with unusual morphology are marked with white arrows. c trypan blue stained a. thaliana leaves at dpi. d quantification of trypan blue stained primary hyphae and secondary hyphae at dpi. e appressoria of wt and vir- on artificial hydrophobic surface. appressoria with unusual morphology are marked with arrows. scale bars = 20 μm. f vegetative growth of wt and vir- on oatmeal agar, potato dextrose agar and czapek-dox minimal medium. g, h average conidial cell volume g and conidial titer h of wt and vir- strains recovered from oatmeal agar after 7 days. the data of separate plates is given. error bars are represented as the standard error from three replicates. significant differences based on t-tests are marked with asterisks when inoculated on potato dextrose agar or minimal medium plates, vir- produced slightly smaller colonies than the wild-type. furthermore, vir- did not show different sensitivity to several stress inducing compounds than the wild-type. in addition to its virulence phenotype, mutant vir- generated less conidia on oatmeal agar plates, which were significantly smaller than wild-type conidia. taken together, the t-dna insertion mutant vir- is severely impaired in virulence due to defects during appressoria differentiation and host cell penetration. vir- encodes a hypomorphic allele of chmob2 in order to identify the mutation responsible for the phenotype of vir- we initially analyzed a t-dna insertion site found in the original mutant screen which suggested the presence of two linked t-dna insertions on supercontig close to ch063_ encoding a homolog to yeast mob. by pcr analyses using a series of primers specific to that locus, both regions flanking the insertion could be isolated and were sequenced. the t-dna insertion site was located upstream of the atg in gene ch063_ and was accompanied by a small genomic deletion of 11 bp. two t-dna copies were inserted in a head to head fashion. attempts to amplify the junction between the two t-dna fragments by pcr were not successful, presumably due to the inverted arrangement of the sequences. the presence of two t-dnas at this genomic context is consistent with the results of previous southern blot analysis probed with a hygromycin resistance dna fragment. ch063_ encodes a predicted aa protein closely related to yeast mob because the t-dna insertion was close to the annotated start codon and as the annotated n-terminus of ch063_ did not match the n-terminus of the yeast protein, it was important to verify the annotated gene model. in order to identify the correct coding region and complete transcript, we performed 5’- and 3’-race pcr using total wild-type rna as template. as shown in fig. 2a, the resulting revised gene model for the chmob gene consists of two 49 nt introns in the coding region, a 5’-utr of 164 nt, a 3’-utr of 216 nt and 834 nt of coding sequence. accordingly, the t-dna insertions of vir- are located 161 bp upstream of the chmob coding sequence. interestingly, the putative chmob transcription start at - as determined by 5’-race pcr, is located in the small genomic sequence which was replaced by t-dna sequence in the genome of vir- since the insertion is positioned upstream of chmob and deletes the putative transcription start, we analyzed chmob mrna accumulation in rna samples from vir- and wild-type c. higginsianum by qualitative and quantitative pcr. in wild-type samples, chmob rna was detected in conidia, in appressoria formed on artificial surfaces, in mycelium and in infected a. thaliana leaves. rna levels reached 20% relative to the housekeeping gene chtubulin-α in appressoria and 55% in rna from a. thaliana leaves 4 days after infection with wild-type c. higginsianum. chmob was expressed strongly during appressoria formation as early as 4 h after inoculation. in contrast, chmob transcript levels were reduced more than ten-fold in all vir- samples. chmob expression in vir- infected a. thaliana could not be analyzed because virulence of this strain was too limited.fig. vir- encodes a hypomorphic chmob allele. a schematic representation of the chmob locus in wild-type and vir-. beginning and end of the chmob transcript are shown relative to the start codon. introns are depicted as striped boxes. lb = left border; rb = right border. b quantitative rt-pcr of chmob mrna abundance in conidia, appressoria, mycelium and in infected plants. the data were normalized against α-tubulin and are shown as the average of three biological replicates. c semi-quantitative rt-pcr of chmob and α-tubulin in rna samples from in vitro appressoria and 12 h after inoculation. cdnas from wt, vir- and Δchace strains were used as template for pcr. genomic wt dna or h2o were used as controls. d, e a. thaliana leaves at 3 days after infection with wt, vir- and vir- strains complemented with either the chmob wild-type allele or with a ptef-mob2-gfp construct. typical trypan blue stained leaves are shown in d. quantifications are shown in e. scale bars = 20 μm. f average volume of conidia from the indicated strains recovered from oatmeal agar after 7 days. the data from separate plates is given. error bars are represented as the standard error from three replicates. significant differences based on t-tests are marked with asterisks in order to verify that the insertion upstream of chmob and the resulting strong reduction of chmob expression is responsible for the phenotype of the vir- mutant, we complemented vir- both with a plasmid harboring the chmob wild-type allele from - to + and with a translational chmob2-gfp fusion under control of the strong and constitutive elongation factor alpha promoter. after reintroduction of wild-type chmob by atmt with pmob virulence was fully restored. when conidia were recovered from oatmeal plates, their cell titers and average cell size were restored to nearly wild-type levels. transformation with ptef1-mob2-gfp fully rescued the conidia cell size phenotype, while conidiation was restored to 80% of wild-type, similar to strains complemented with the wild-type allele. in the ptef1-mob2-gfp transformants, virulence appeared to be even increased relative to the wild-type possibly due to overexpression of chmob in all cases, empty vector controls did not affect the vir- phenotypes. since all observed phenotypes of vir- can be complemented by reintroduction of chmob the phenotype of this mutant must be linked to the identified insertion. in summary, vir- encodes a hypomorphic chmob allele expressing less than 10% mrna, which was generated by insertion of t-dna in the 5’-utr separating the coding region from its promoter. the c. higginsianum genome encodes three members of the mob1/phocein protein family mob belongs to the mob1/phocein protein family, which is widely conserved among eukaryotes and many of its members have been shown to bind and activate ndr kinases. in contrast to metazoans, which contain up to six different mob proteins, fungal genomes contain between two and four mob coding genes. the resulting proteins can be grouped into three clades: the first contains proteins with high similarity to yeast mob which binds the dbf kinase, while the second clade clusters together with yeast mob which binds to cbk. the third clade contains phocein, the founding member of the mob1/phocein family from rattus norvegicus, and proteins from filamentous fungi like s. macrospora or n. crassa mob which have been reported to be essential for development of reproductive protoperithecia and for hyphal fusion. all filamentous fungi analyzed in fig.  contain one protein from each clade, except s. macrospora and n. crassa, which both harbor two mob2-like genes. in n. crassa, mob2a and mob2b have partly overlapping functions and were both shown to interact with the cbk like kinase cot. as c. higginsianum contains one protein from each clade, we assume that each mob protein has functions similar to the respective homologs from its clade.fig. the genome of c. higginsianum encodes three mob-like proteins. unrooted phylogenetic tree of mob proteins from c. higginsianum and other fungi. the tree was generated with the geneious tree builder plugin using a blosum cost matrix and the jukes-cantor tree building method. the founding member of the mob family from rat was included as a reference. branch lengths correspond to substitutions per site. scale bar =  substitutions per site chmob and chcbk may be essential genes in c. higginsianum in order to obtain Δchmob null mutants, we attempted to perform targeted gene knockout of chmob by transformation of the non-homologous end-joining defective mutant Δchku with the chmob deletion plasmid pck this plasmid harbors the hygromycin resistance cassette flanked by chmob upstream and downstream sequence on the t-dna. in the Δchku background >90% of all transforming dna should integrate by homologous recombination. in three independent experiments, however, we recovered only transformants that harbored ectopically integrated dna. in contrast, transformations of the nhej proficient wild-type strain yielded > transformants. we therefore expect that chmob may be an essential gene in c. higginsianum, at least in the Δchku background. interestingly, mob can be inactivated in s. cerevisiae and in n. crassa. as described for s. cerevisiae, mob family proteins often act as co-activators for ndr/lats kinases. in particular, mob binds to cbk kinase in yeast. we assumed that chmob has a similar function in c. higginsianum and may act through activation of chcbk we attempted to delete chcbk in the Δchku background. in three independent experiments this failed similar to chmob. it is therefore likely that both chmob and chcbk have essential functions in c. higginsianum. in addition, attempts to generate knock-down mutants of these genes by expression of antisense-rnas did not lead to transformants with significantly reduced levels of the respective transcripts. chmob co-localizes with the potential kinase chcbk in appressoria in order to further examine the function of c. higginsianum mob we investigated the subcellular localization of chmob and its potential binding partner chcbk we constructed a strain where the genomic copy of chcbk was replaced by a cbk1-mcherry fusion and where chmob was simultaneously replaced by mob2-gfp. the resulting strain had no significant defects in either virulence, conidiation or vegetative growth except for producing slightly smaller conidia. since both chmob and chcbk may be essential in c. higginsianum, reduced chmob or chcbk function should have obvious effects. we therefore concluded that both fusion proteins were functional. in conidia isolated from oma plates, both fusion proteins were more or less evenly distributed over the whole cell except for the nucleus, which appeared to be partly excluded. the two proteins co-localized to some extent. early upon in vitro appressoria formation, when appressoria were unmelanized, both cbk1-mcherry and mob2-gfp were completely overlapping. in the germinated conidia attached to these appressoria, however, the fusion proteins seemed to be localized to different compartments. in mature appressoria and their corresponding germinated conidia, both fusion proteins were again co-localized all over the cells except in nuclei, where both were absent. in axenic cultures, co-localization could not be addressed because the cbk1-mcherry fusion was barely detectable. in planta, we could not detect either of the two fusion proteins, presumably because of the high background fluorescence in green leaves. taken together, mob2-gfp and cbk1-mcherry show the same subcellular localization during appressoria formation.fig. chmob co-localizes with chcbk in the cytoplasm. conidia of Δchcbk1:: cbk1-mcherry Δchmob2:: mob2-gfp were rinsed off oatmeal plates and analyzed by clsm during in vitro appressoria formation. a conidia, b in vitro ap at hpi, c in vitro ap at hpi. bright field, gfp and mcherry channels are shown from left to right. the rightmost picture shows an overlay of gfp and mcherry channels. the intensities between panels are not directly comparable, because different settings were used to optimally visualize protein localization. scale bars = 20 μm mob2-gfp physically interacts with cbk1-ha chmob and chcbk can occupy the same subcellular compartment. consequently, they might physically interact with each other during certain times in the cell cycle. in order to test their possible interaction, we performed co-immunoprecipitation of chmob and chcbk we constructed a strain where chcbk was fused to a 6xha tag at its own genetic locus. in addition, this strain ectopically expressed mob2-gfp under control of the tef1a promoter. both proteins could be detected in axenic mycelium after western blotting with anti-gfp and anti-ha antibodies. mob2-gfp was detected at about 60 kda. cbk1-ha produced several bands between and 95 kda, possibly because of different phosphorylation states of the protein or due to protein degradation. we used protein extracts from mycelium to investigate the interaction of chcbk with chmob first, cbk1-ha was immunoprecipitated with anti-ha antibodies coupled to magnetic beads. the successful pull down of cbk1-ha is shown in the western blot decorated with anti-ha antibodies. the presence of mob2-gfp in these immunoprecipitates was determined by decorating the western blots with anti-gfp antibodies. in extracts containing both fusion proteins, mob2-gfp was efficiently co-immunoprecipitated with cbk1-ha, but not from control extracts lacking cbk1-ha. similarly, when mob2-gfp was pulled down with anti-gfp antibody coated beads, cbk1-ha was co-precipitated. some cbk1-ha bands were missing from the anti-gfp ip, suggesting that at least some of the chcbk forms might not bind to chmob the successful co-precipitation of chmob and chcbk indicates that the two proteins can interact in axenic c. higginsianum cells. while technically not testable, it is likely that this interaction also occurs during infection.fig. chmob interacts with chcbk whole cell extracts from strains expressing different combinations of chmob2-gfp and chcbk1-ha were used to analyze the interaction of chmob and chcbk by immunoprecipitation. strains used from left to right: Δchcbk1:: cbk1-ha ptef1a-mob2-gfp, the parental strain Δchku, Δchcbk1:: cbk1-ha and ptef1a-mob2-gfp. western blots of whole cell protein extracts, immunoprecipitates with anti-ha antibody coated beads and immunoprecipitates with anti-gfp antibody coated beads were decorated either with anti-gfp or with anti-ha primary antibodies. the band corresponding to mob2-gfp is marked with an arrow. the multiple bands corresponding to cbk1-ha are marked with a bracket. asterisks mark bands corresponding to primary antibody which are present in the ip and therefore are recognized by the secondary antibody next, we tried to verify this interaction by mass spectrometry using cbk1-ha expressing cell extracts without overexpressing mob2-gfp. this approach could potentially also identify downstream targets of chcbk1-phosphorylation and could show whether or not chmob is the sole activator of chcbk we pulled down cbk1-ha from extracts of axenic mycelium of cy and from extracts of the parental strain as control in two biological replicates using anti-ha antibody-coated magnetic beads. the eluates were analyzed by nano lc-ms/ms and quantified label-free by integrating peak areas. chmob was only identified in the cbk1-ha ips but not in samples from the untagged parental strain, further verifying the interaction of these two proteins in c. higginsianum. the other peptides identified were probably contaminants as they consisted mainly of highly expressed enzymes from the citrate cycle, from glycolysis or proteins associated with ribosomes. the lack of potential chcbk targets in the pull-downs is likely due to transient interactions of the kinase with its substrates. alternatively, the important proteins may not be present or not targeted by chcbk in samples of in vitro mycelium. two potential downstream targets of the chmob2/chcbk complex are required for full pathogenicity two well described cbk targets in yeast are the transcription factor ace and the mrna-binding protein ssd. we therefore tested whether or not their orthologs from c. higginsianum are involved in pathogenicity. we identified possible orthologs in the c. higginsianum genome. the closest homolog to scace was ch063_ with % identity. after comparison with the sequence of s. cerevisiae ace and the more closely related aface of a. fumigatus, we concluded that the automatic annotation of ch063_ was not complete and extended the n-terminus by amino acids resulting in a revised c. higginsianum chace gene model encoding for amino acids. this extension could be verified by sequencing chace cdna. the revised protein has % sequence identity to scace and % to aface over the full sequence. the region containing the c2h zinc finger dna binding domain showed % sequence identity to aface the potential c. higginsianum ssd shows %, % and % sequence identity to the potential orthologs from saccharomyces cerevisiae, magnaporthe grisea and colletotrichum lagenarium, respectively. although both chssd and chace show less than 40% identity to their respective yeast homologs, they are clearly the best match in reciprocal blast searches. we generated Δchace and Δchssd deletion strains in the Δchku background and examined their phenotype. consistent with a role of chace and chssd downstream of the chmob2/chcbk complex, both mutants exhibited significantly reduced virulence. furthermore, Δchace and Δchssd strains showed decreased vegetative fitness, as their colonies on minimal medium were smaller by about 35% and 50% than wild-type, respectively. none of the mutants showed significantly altered sensitivity to osmotic stress, heat, h2o or cell wall stress. interestingly, conidial cell size and especially the conidial titer recovered from oma plates were reduced in the Δchace mutant similar to the phenotype observed in vir- the phenotype of Δchace could be rescued by reintroduction of wild-type chace into the Δchku locus of this strain, further confirming the involvement of chace in virulence and conidiation. the decrease in fitness of the Δchace and Δchssd mutants could contribute to their virulence phenotype. the conidiation phenotype of the Δchace mutant, however, is likely to be a specific phenotype, as slow growing mutants do not a priori produce less conidia. it was not possible to generate a Δchace Δchssd double mutant.fig. chace and chssd are required for full virulence of c. higginsianum. a schematic representation of the genomic loci of chace and chssd and the knockout cassettes of the respective deletion plasmids. the position of 5’ and 3’ flanking regions are given relative to the respective start codon. b spray infection of a. thaliana at dpi. primary hyphae and secondary hyphae produced by the indicated strains. c vegetative growth of the indicated strains on potato dextrose agar and czapek-dox agar minimal medium. d average number of conidia and cell volume for the indicated strains recovered from oatmeal agar after 7 days. the data from separate plates is shown. the strains used in b-d were wild-type, vir- Δchace, Δchssd and the Δchku parental strain of the last two. error bars are represented as the standard error from three replicates. significant differences based on t-tests are marked with asterisks in order to analyze if chace is regulated by the chmob2/chcbk complex in c. higginsianum as in s. cerevisiae, we analyzed the mrna amount of potential orthologs of the scace targets cts and scw and of the s. pombe ace target mid in samples from vir- and Δchace we expected that if chace activity were regulated by chmob the transcript amount of chace targets might be different in vir- which expresses only little chmob, and in Δchace mutants, which lacks chace however, using both semi-quantitative and quantitative rt-pcr we could not detect significant chcts chscw or chmid transcript changes in appressoria samples in comparison to the wild-type. the mrna level of chace was not significantly different between vir- and the wild-type. this could indicate that similar to the situation in yeast, chace is not regulated on the level of mrna accumulation but by chcbk1-dependent phosphorylation. furthermore, Δchcts strains, which lack one of the genes predicted to encode chitinase, did not show any defects in virulence, conidiation or cell separation. a cell separation phenotype was observed in yeast Δcts mutants. this may indicate that ace regulates different targets in c. higginsianum than it does in yeasts. in summary, we were not able to identify downstream targets of chmob2/chcbk in c. higginsianum, but deletion of two cbk targets known from other systems resulted in pathogenicity and conidiation phenotypes resembling those of vir- the roles of chmob and chmob in c. higginsianum while mob and mob seem to be present in all fungal genomes, mob is only found in filamentous fungi and higher eukaryotes. the mammalian ortholog of mob was identified in rat dendritic cells and has been shown to bind to striatin, a calmodulin binding protein. in fungi, mob has been reported to be essential for sexual development and for hyphal fusion in n. crassa and s. macrospora. although c. higginsianum has not been described to perform a sexual cycle, a potential ortholog of this gene is encoded in the c. higginsianum genome. we therefore investigated the potential roles of chmob in c. higginsianum by targeted gene knockout of chmob. except for a very mild reduction in colony size on both pda and minimal medium, we did not observe significant changes either in virulence or conidiation of the resulting Δchmob mutants. the expression pattern of chmob did not give any further hint regarding its physiological function, as it was expressed at low, constitutive levels under all tested conditions. we cannot exclude that chmob has a specific function under different, specific conditions. in yeasts and n. crassa, mob and mob have distinct, but similar functions, namely the binding and activation of a ndr/lats-kinase. in s. cerevisiae, mob binds to cbk in the ram pathway and mob binds to dbf in the mitotic exit network. although binding of mob or mob to their respective kinases is thought to be specific, there may be crosstalk between men and ram networks in s. cerevisiae. we therefore investigated if chmob and chmob have overlapping functions in c. higginsianum by genetically inactivating chmob. transformation of Δchku with a chmob deletion plasmid resulted in mutants with a strong growth phenotype on all tested media. in addition, these mutants showed strong sensitivity to the cell wall stress inducing and chitin binding dye congo red, indicating a defect in chitin turnover in the cell wall. furthermore, Δchmob mutants hardly produced any conidia on oatmeal medium. the few conidia that could be recovered were smaller by about 50% and were significantly reduced in formation of appressoria, primary hyphae and secondary hyphae after droplet inoculations. in contrast to wild-type conidia which form a septum during germination, Δchmob conidia were never septated. furthermore, Δchmob conidia were often connected to or other conidia, possibly because they did not undergo proper cell separation during conidiation. hyphae from axenic mycelium of Δchmob strains also hardly produced any septae at all, in contrast to the parental strain. the strong septation phenotype of Δchmob strains is similar to mutants in s. cerevisiae and s. pombe, where mob is required for completion of mitosis, initiation of cytokinesis and septation.fig chmob is required for septum formation and cell division. two independent Δchmob transformants and the parental strain Δchku were tested for vegetative growth, virulence and the ability to produce septae. a vegetative growth on oatmeal agar, potato dextrose agar and czapek-dox minimal medium. b a. thaliana 4 days after droplet inoculation. representative images of trypan blue stained leaves are shown on the right. the quantification of appressoria, primary hyphae and secondary hyphae of this infection is shown on the left. the data of plants per strain with leaves per plant with at least counted appressoria per leaf is given. error bars are represented as the standard error from three replicates. significant differences based on t-tests are marked with asterisks. c axenic mycelium stained with calcofluor white. septae are marked with white arrows. scale bars = 20 μm in summary, c. higginsianum contains two additional mob genes. consistent with a function of mob during sexual development in other systems, deletion of chmob did not lead to any significant phenotype in c. higginsianum which is lacking a sexual cycle. in contrast, knockout of chmob leads strong defects in appressoria formation, vegetative growth, septation, conidiation and infection. discussion screening for genes involved in pathogenicity though aimed at identification of genes with functions exclusively required for certain aspects of the infection process, will always also identify genes with vegetative functions at many stages of their life cycle. atpases, intracellular transport, nutrition and morphogenesis are notable examples. appressorial pathogens like colletotrichum higginsianum undergo switches in polarity during infection of plant cells. in particular, the directed formation of a penetration peg underneath the appressorium is essential for infection. it therefore is not too surprising to identify polarity determinants like mob in a screen for pathogenicity genes. we identified a weak allele of chmob in the vir- mutant that in most cases produces appressoria with morphologic defects like elongation or secondary outgrowths. these appressoria are not able to penetrate and to initiate the infection process. since a fraction of the vir- appressoria looked normal and appeared functional, the mutant shows residual weak symptoms in some spots on the leaf, resulting in a mutant with reduced pathogenicity. in addition, vir- produces less and smaller conidia. although vir- expresses less than 10% of chmob mrna relative to the wild-type, it is not a null mutant. since the t-dna insertion in the genome of vir- is located just in front of the chmob reading frame, it is likely that residual chmob activity may be present, at least in some cells. the phenotype of a true null mutant could not be analyzed because all attempts to generate a deletion allele by homologous recombination failed. the likely explanation, therefore, is that chmob is an essential gene at least in the Δchku background used for all knockouts. a low expression, which may vary from cell to cell, could also be the reason why not all mutant cells behave identical, resulting in incomplete penetrance of the underlying genetic defect. while this makes interpretation of the phenotype more difficult, the fortuitous isolation of a weak allele allowed us to characterize the functions of chmob the defect of most vir- appressoria to penetrate the host tissue correlated with morphological defects. in agreement with a possible essential function for chmob its binding partner, the ndr/lats kinase chcbk could also not be knocked out. while a failure to generate knockout mutants is no evidence for an essential function, it resembles the situation in yeast and s. pombe, where mob and cbk have been found to be essential.. interestingly, the lethality of ram mutations in yeast is suppressed by loss of ssd, showing that the consequences of ram mutations may be strain specific. in most systems, mob and cbk mutations are indeed associated with pleiotropic phenotypes and morphological defects. in the opportunistic human pathogen c. albicans, all mutants in the ram pathway including camob and cacbk are viable but show hyperpolarization. in the basidiomycetes c. neoformans and u. maydis, mutants of the cbk ortholog ukc mob and all other associated proteins thought to be involved in the same pathway lead to hyperpolarized growth and decreased virulence. in a. nidulans, mutants in cbk and mob orthologs show growth defects, strongly reduced ability to form conidia, altered conidia cell size and increased number of nuclei in spores. in n. crassa, which is more closely related to c. higginsianum, the cot- mutant and the mob2a mob2b double mutant show increased rate of branching in axenic mycelium, reduced growth rate and reduced conidiation. most phenotypes observed in our study for the chmob mutant vir- are consistent with functions reported for mob and cbk in filamentous fungi, including a function in conidiation. reduced cell size of conidia was an obvious phenotype of vir- altered conidial cell size may be the consequence of altered cell division or septation in conidial precursor cells. altered conidial cell size was also observed in n. crassa cot mutants. they were, however, times larger than wild-type cells, rather than smaller as in the case of c. higginsianum. we also observed about 50% reduction in conidiation ability in the vir- mutant. considering that vir- is not a null allele of chmob this phenotype is reminiscent of a. nidulans and n. crassa mob mutants which largely fail to form conidia. conidiation is critically dependent on septation, which in turn is also dependent on the mob1/dbf2-dependent men or sin signalling pathways in many fungi. in fact, n. crassa and a. nidulans Δmob show sporulation defects similar to the c. higginsianum Δchmob mutants described in this study. it is possible that the mob1/dbf and mob2/cbk complexes have overlapping functions or that they control each other. the latter may be the case in related systems. in n. crassa, mob appears to affect cot activity more than its direct interactor and co-activator mob2b. in s. pombe, spsid controls sporb activity, while mob and cdc activity is required for proper mob and ace localization in yeast. for comparison, a simplified model of the potential interplay between men and ram of s. cerevisiae is shown in additional file 10: figure s in addition to a strong conidiation phenotype, Δchmob mutants exhibited attenuated ability to form appressoria, primary and secondary hyphae upon infection and showed a severe septation defect. these phenotypes are similar to the corresponding mutants in n. crassa and a. nidulans. mob is essential both in s. cerevisiae and s. pombe, but cells harboring conditional alleles of mob either arrest in late mitosis or, in the case of s. pombe, do not septate and become multinucleate. in addition to nuclear division, s. cerevisiae mob also seems to have a role during cytokinesis and cell separation. based on these similarities, it is likely that the function of mob and its associated signalling pathway is conserved among ascomycetes. what are the critical targets of the mob2/cbk pathway in filamentous fungi? similar to the situation in yeast, it was found that deletion of the n. crassa homolog of ssd can suppress defects of the cot allele suggesting that ssd is an important component of the ram pathway also in filamentous fungi. in s. cerevisiae, ssd inactivation is thought to occur through cbk phosphorylation. because ssd may be a conserved cbk target we analyzed the phenotype of a Δchssd mutant allele. we found that the lack of chssd leads to attenuated virulence in c. higginsianum. the effect on virulence was similar to vir- and to Δssd mutants in the plant pathogens c. lagenarium and magnaporthe grisea supporting the notion that ssd is a conserved virulence associated target of the ram pathway. in c. lagenarium, Δssd mutants mostly failed to penetrate and intracellular hyphae were only detected very rarely. in m. grisea, Δssd mutants were still able to produce intracellular primary hyphae on rice plants, but they were mostly restricted to dead host cells. we also analyzed the effect of chace on pathogenicity in c. higginsianum because it may be a conserved target of the ram pathway. again, we saw a reduction of pathogenicity and, interestingly, a comparable effect on cell volume in conidia and reduced conidiation. however, we found no evidence for posttranslational regulation of chace by the chmob2/chcbk complex, as described for s. cerevisiae. we observed that c. higginsianum mob and cbk localize to the cytoplasm and are excluded from nuclei in conidia and during in vitro appressoria formation. in baker’s yeast, however, mob and cbk proteins can be found at sites of polarized growth like the cortex of the growing bud and the mating projection or in the daughter cell nucleus and the septum during cell separation, where they supposedly regulate cytokinesis genes through the transcription factor scace. this difference in localization could indicate that ace is not a conserved mob2/cbk target in filamentous fungi. in the absence of verified targets for the transcription factor chace it also remains unclear whether or not chace and yeast ace have a common function. a protein homologous to scace was also analyzed in a. fumigatus, where knockout mutants had attenuated ability to produce conidia, abnormal cell wall architecture and were hypervirulent in a mouse model. this protein is more closely related to chace than to the yeast protein and, given their common conidiation phenotype, they may be orthologous gene products.  this study showed that the mob-family protein chmob from the plant pathogen colletotrichum higginsianum is involved in virulence on arabidopsis thaliana and has a role in conidiation. chmob forms a complex with the conserved chcbk kinase. the study further analyzed the functions of the other two mob proteins encoded in c. higginsianum by targeted gene knockouts. chmob is required for conidiation, cytokinesis and plant infection, while Δchmob mutants have no obvious phenotype in vegetative cells or during infection. 
we identified a hypomorphic allele of chmob in a random insertional mutant of the hemibiotrophic ascomycete fungus colletotrichum higginsianum. the genome of c. higginsianum encodes two more mob proteins. this study shows that chmob binds to the conserved ndr/lats kinase chcbk and plays an important role in pathogenicity of colletotrichum higginsianum on arabidopsis thaliana. phytopathogenic ascomycete fungusatmtcolletotrichum higginsianumconidiationndr kinase cbk1mob1mob2mob3ace2ssd1cts1issue-copyright-statement© the author 2017 mob family proteins are conserved between animals, plants and fungi and are essential for the activation of ndr kinases that control crucial cellular processes like cytokinesis, proliferation and morphology. the mutant is impaired in conidiation, host penetration and virulence on arabidopsis thaliana.
 mob family proteins are conserved between animals, plants and fungi and are essential for the activation of ndr kinases that control crucial cellular processes like cytokinesis, proliferation and morphology.  we identified a hypomorphic allele of chmob in a random insertional mutant of the hemibiotrophic ascomycete fungus colletotrichum higginsianum. the mutant is impaired in conidiation, host penetration and virulence on arabidopsis thaliana. chmob binds to and co-localizes with the ndr/lats kinase homolog chcbk mutants in the two potential chcbk downstream targets chssd and chace show defects in pathogenicity. the genome of c. higginsianum encodes two more mob proteins. while we could not detect any effect on pathogenicity in Δchmob mutants, chmob is involved in conidiation, septae formation and virulence.  this study shows that chmob binds to the conserved ndr/lats kinase chcbk and plays an important role in pathogenicity of colletotrichum higginsianum on arabidopsis thaliana. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords phytopathogenic ascomycete fungusatmtcolletotrichum higginsianumconidiationndr kinase cbk1mob1mob2mob3ace2ssd1cts1issue-copyright-statement© the author 2017 