Our mission: Apply in vivo approaches to study RNA Biology in Ustilago maydis
Movie 1 The RNA-binding protein Rrm4 shuttles bidirectionally in filaments of U. maydis
The plant pathogen Ustilago maydis is the causative agent of corn smut disease. Prerequisite for infection is a drastic morphological switch from yeast-like to filamentous growth. RNA-binding proteins are important regulators of this developmental program. During recent years we have focused on two aspects:
- the role of Rrm4 during microtubule-dependent mRNA transport
- the role of Khd4 during regulation of morphology and pathogenicity
Infectious filaments grow with a distinct axis of polarity. They expand at the apical pole and insert retraction septa at the basal end, confining the cytoplasm to the tip compartment. This growth mode leads to formation of regularly spaced empty sections at the rear end of filaments. Loss of rrm4 causes the formation of aberrant filaments and reduced virulence. The majority of filaments grows bipolar and hardly any retraction septa are formed (Fig. 1).
Fig. 1 Loss of Rrm4 results in disturbed filamentous growth. (A) Filament of laboratory strain AB33 expanding at the apex (right) and inserting retraction septa at the basal pole (left). This leads to the formation of empty sections. (B) Deletion of rrm4 causes no aberrant growth phenotype in yeast form. (C) Loss of Rrm4 leads to bipolar growth. The initial cell (center) switches to filamentous growth by expanding at both ends (size bar = 10 µm). Pictures taken from Vollmeister et al 2012 FEMS Microbio Rev
The key RNA-binding protein contains three N-terminal RRMs (RNA recognition motif) and a C-terminal PABC (poly[A]-binding protein C terminus). The latter domain is predicted to function in protein-protein interaction. The protein shuttles bidirectionally along microtubules (Movie 2).
Movie 2 The RNA-binding protein Rrm4 shuttles along microtubules
In order to identify target mRNAs we applied in vivo UV crosslinking (CLIP). Rrm4 was found to interact with distinct sets of mRNAs including those encoding the small GTPase Rho3 and the ubiquitin fusion protein Ubi1. To verify these results we used RNA live imaging to prove that target mRNAs shuttle bidirectional along microtubules in particles that co-localise with Rrm4 (Movie 3).
Movie 3 RNA live imaging of ubi1 mRNA in infectious filaments
Deletion of the RNA-binding domains of Rrm4 verified that target mRNA transport is lost in the absence of RNA binding. However, the remaining part of the protein still shuttled along microtubules. This indicated that Rrm4 does not only hitchhike along mRNAs, but forms an integral part of the transport unit.
The small G protein Rho3, which is encoded by an Rrm4 target mRNA, accumulates at the retraction septa of filaments (Fig. 2). These observations suggest that also in fungi long-distance transport of mRNA is important to promote the defined subcellular localisation of encoded proteins. Such mRNA transport processes along microtubules are so far only known from higher eukaryotes like embryos of Drosophila melanogaster and mammalian neurons.
Investigating the mechanism of transport revealed that mRNPs are co-transported with Rab5a-positive endosomes by the action of Kinesin-3 type Kin3 and split dynein Dyn1/2 (Fig. 2). This constitutes a novel function of endosomes.
Fig. 2 mRNP trafficking during formation of infectious filaments in U. maydis. Bidirectional mRNA shuttling is mediated by co-transport on endosomes. Pictures taken from Vollmeister et al 2012 RNA Biol.
In the future we would like to identify the composition of the mRNP particles, establish how mRNPs are associated with endosomes, and elucidate the connection between disturbed polarity and reduced pathogenicity.
The role of Khd4 during regulation of morphology and pathogenicity
Deletion of khd4, encoding an RNA-binding protein with five K homology (KH) domains, causes aberrant cell morphology and reduced virulence (Fig. 3).
Recently, we demonstrate using the yeast three hybrid system that Khd4 recognises the sequence AUACCC in vivo via its tandem KH domains 3 and 4. This sequence functions most likely as regulatory RNA element in U. maydis, since bioinformatic analyses reveal that it accumulates in 3' untranslated regions. Consistently, an independent transcriptional profiling approach revealed that the binding motif is significantly enriched in transcripts differentially regulated in khd4 deletion strains (Fig. 4).
Since the vast majority of potential Khd4 target mRNAs exhibit increased expression in deletion mutants, Khd4 might promote mRNA instability. Mutants that fail to bind AUACCC resemble deletion mutants exhibiting altered cell morphology, disturbed filamentous growth and severely reduced virulence. Hence, RNA binding is essential for function of Khd4 stressing the importance of posttranscriptional control in regulating morphology and pathogenicity. In the future, our emphasis will be to uncover which targets of Khd4 are responsible for abnormal cell morphology and reduced pathogenicity.