The RNA-binding protein Rrm4 is essential for polarity in Ustilago maydis and shuttles along microtubules.

Formation of polar-growing hyphae is essential for infection by the plant pathogen Ustilago maydis. Here we observe that loss of RNA-recognition motif protein Rrm4 caused formation of abnormal hyphae. The insertion of septa at the distal pole was abolished and a significantly increased number of hyphae grew bipolarly. UV-crosslinking experiments revealed that Rrm4 bound RNA via its N-terminal RRMs and that its RNA-binding activity was substantially increased during filamentation. Rrm4 assembled into particles that shuttled bidirectionally along microtubules to both poles. Recruitment of Rrm4 into particles increased during filamentation, and mutations in the peptide-binding pocket of its PABC domain caused abnormal particle formation as well as polarity defects. Shuttling was mediated by active transport because loss of conventional kinesin, which interferes with the balance of microtubule-dependent motors, caused accumulation of particles at the poles resulting in disturbed polarity. Thus, constant transport of the RNA-binding protein towards the poles is needed to orchestrate hyphal growth. Since a mutation of the N-terminal RRM that leads to reduced RNA binding in vivo also affected polarity, Rrm4 might regulate polarity of the infectious hyphae by transporting RNA from the nucleus to cell poles.

Isolation of a novel ABC-transporter gene from soybean induced by salicylic acid.

This paper reports on the identification and characterization of a new ATP-binding cassette (ABC) transporter which was identified as a salicylic acid-induced gene from soybean (Glycine max cv. Williams 82) in a subtractive suppression hybridization approach. A fragment of an ABC-transporter gene was used to isolate a full-length cDNA clone for this gene with a length of 4750 bp. The encoded protein has a length of 1447 amino acids and is composed of two similar repeat units typical of full-size ABC transporters. The sequence displays a close relationship to plant pleiotropic drug resistance (PDR)-type transporters and, on a homology basis, clusters together with the Arabidopsis thaliana PDR12 gene, suggesting GmPDR12 as a name for the gene isolated from soybean. GmPDR12 is rapidly responsive to salicylic acid and methyl jasmonate. The mRNA starts to accumulate 30 min after the addition of the signalling compounds. Salicylic acid is required for the execution of the hypersensitive reaction in soybean cell suspension cultures inoculated with Pseudomonas syringae pv. glycinea. It has been demonstrated previously that salicylic acid can be substituted by a variety of functional analogues of salicylic acid. All of these compounds lead to a strong and rapid transcriptional activation of GmPDR12, suggesting a shared signalling pathway.

Role for RNA-binding proteins implicated in pathogenic development of Ustilago maydis.

Ustilago maydis causes smut disease on corn. Successful infection depends on a number of morphological transitions, such as pheromone-dependent formation of conjugation tubes and the switch to filamentous dikaryotic growth, as well as different types of mycelial structures during growth within the host plant. In order to address the involvement of RNA-binding proteins during this developmental program, we identified 27 open reading frames from the genome sequence encoding potential RNA-binding proteins. They exhibit similarities to RNA-binding proteins with Pumilio homology domains (PUM), the K homology domain (KHD), the double-stranded RNA binding motif (DSRM), and the RNA recognition motif (RRM). For 18 of these genes, we generated replacement mutants in compatible haploid strains. Through analysis of growth behavior, morphology, cyclic AMP response, mating, and pathogenicity, we identified three candidates with aberrant phenotypes. Loss of Khd1, a K homology protein containing three KHDs, resulted in a cold-sensitive growth phenotype. Deletion of khd4 encoding a protein with five KHDs led to abnormal cell morphology, reduced mating, and virulence. rrm4Delta strains were affected in filamentous growth and pathogenicity. Rrm4 is an RRM protein with a so far unique domain organization consisting of three N-terminal RRMs as well as a domain found in the C terminus of poly(A)-binding proteins. These results indicate a role for RNA-binding proteins in regulation of morphology as well as in pathogenic development in U. maydis.