Abstract
LIM domain proteins contain contiguous double-zinc finger domains and play important roles in cytoskeletal re-organisation and organ development in multi-cellular eukaryotes. Here, we report the characterization of four genes encoding LIM proteins in the rice blast fungus Magnaporthe oryzae. Targeted gene replacement of either the paxillin-encoding gene, PAX1, or LRG1 resulted in a significant reduction in hyphal growth and loss of pathogenicity, while deletion of RGA1 caused defects in conidiogenesis and appressorium development. A fourth LIM domain gene, LDP1, was not required for infection-associated development by M. oryzae. Live cell imaging revealed that Lrg1-GFP and Rga1-GFP both localize to septal pores, while Pax1-GFP is present in the cytoplasm. To explore the function of individual LIM domains, we carried out systematic deletion of each LIM domain, which revealed the importance of the Lrg1-LIM2 and Lrg1-RhoGAP domains for Lrg1 function and overlapping functions of the three LIM domains of Pax1. Interestingly, deletion of either PAX1 or LRG1 led to decreased sensitivity to cell wall-perturbing agents, such as Congo Red and SDS (sodium dodecyl sulfate). qRT-PCR analysis demonstrated the importance of both Lrg1 and Pax1 to regulation of genes associated with cell wall biogenesis. When considered together, our results indicate that LIM domain proteins are key regulators of infection-associated morphogenesis by the rice blast fungus.
Highlights
The LIM domain is named after the three proteins (Lin-11, Isl1 and Mec-3) and consists of two tandemly-repeated zinc fingers within a conserved domain of 50-60 amino acids with consensus sequence CX2CX16-23HX2CX2CX2CX16-21CX2(C/H/D), where X denotes any amino acid; and/indicates alternatives [1,2,3,4,5,6,7]
Deletion of either PAX1 or LRG1 led to decreased sensitivity to cell wall-perturbing agents, such as Congo Red and sodium dodecyl sulfate (SDS). Quantitative RT-PCR (qRT-PCR) analysis demonstrated the importance of both Lrg1 and Pax1 to regulation of genes associated with cell wall biogenesis
Previous studies showed that C-terminal LIM domains in paxillin are involved in binding the protein tyrosine phosphatase PTP-PEST to target the protein to focal adhesions, and to bind a- and c-tubulin to direct an interplay between actin filaments and microtubules [20,21,22]
Summary
The LIM domain is named after the three proteins (Lin-11, Isl and Mec-3) and consists of two tandemly-repeated zinc fingers within a conserved domain of 50-60 amino acids with consensus sequence CX2CX16-23HX2CX2CX2CX16-21CX2(C/H/D), where X denotes any amino acid; and/indicates alternatives [1,2,3,4,5,6,7]. Unlike the DNA-binding function of many zinc fingers, the LIM domain does not bind DNA, but instead mediates specific protein-protein interactions, acting as a conserved scaffold to recognize diverse target proteins [5,7,8,9,10]. Group 2 constitutes LMO proteins consisting of two or more LIM domains, but unlike nuclear LMOs, proteins in this group are present in the cytoplasm or nucleus or can shuttle between compartments to regulate gene expression. Group 3 contains paxillin, zyxin, testin and enigma, which possess additional functional domains such as LD (leucineaspartate repeat), ATD (actin-target domain) and PDZ (first letters of three proteins PSD95, Dlg and Zo-1). The conserved Group 3 paxillin proteins in animals consist of four characterized LIM domains at C-termini and an additional five LD motifs at the N-terminus [17,18]. Together with Rho-GTPases and the formin protein AgBni, AgPxl regulates spore length and spore wall integrity by directly interacting with AgRho1a and AgRho1b [30]
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