Biology subjects

Rico-Ramirez, A. M.

Publications and source records attributed to Rico-Ramirez, A. M..

3 recordsLinked to original sources

bioRxiv2025

Construction of a randomly barcoded insertional mutant library in the filamentous fungus Trichoderma atroviride

Filamentous fungi play key roles in ecosystems, agriculture, biotechnology, symbiosis, and disease, yet the large-scale characterization of gene function in these organisms remains limited by low transformation efficiencies and their multinucleate, syncytial cells, which complicate high-throughput screening strategies. To address the challenge of high-throughput screening in filamentous fungi, we developed methods to construct a genome-wide barcoded insertional mutant library in Trichoderma atroviride, a filamentous fungus widely used as a biocontrol agent against bacterial and fungal plant pathogens. Our strategy leveraged randomly barcoded transfer DNA insertions from plasmid libraries containing hundreds of millions of unique DNA barcodes and a broad host-range drug resistance marker delivered via Agrobacterium tumefaciens into T. atroviride. By optimizing transformation conditions, we achieved up to 600 independent transformants per infection event, resulting in a library of over 31,000 mapped insertions disrupting 7,104 of the 11,863 predicted genes in the T. atroviride genome. This resource establishes a scalable platform for high-throughput functional genomics in filamentous fungi, enabling both fundamental investigations of fungal biology and engineering approaches toward improved medical applications, biotechnology, and sustainable agriculture.

systems biology↗

bioRxiv2025

Identification of regions required for allelic specificity at the cell wall remodeling allorecognition checkpoint in Neurospora crassa

Allorecognition is the ability of organisms/cells to differentiate self from non-self. In the fungus Neurospora crassa, allorecognition systems serve as checkpoints to restrict germling/hyphal fusion between genetically incompatible strains. The cell wall remodeling (cwr) checkpoint functions after chemotrophic interactions and is triggered upon cell/hyphal contact, regulating cell wall dissolution and subsequent cell fusion. The cwr region consists of two linked loci, cwr-1 and cwr-2, that are under severe linkage disequilibrium. Phylogenetic analysis of wild N. crassa populations showed that cwr-1/cwr-2 alleles fall into six different haplogroups (HGs). Strains containing deletions of cwr-1 and cwr-2 will fuse with previously HG incompatible cells, indicating cwr negatively regulates cell fusion. CWR-1 encodes a polysaccharide monooxygenase (PMO) domain that oxidatively degrades chitin; the PMO domain is sufficient to cause fusion arrest and confers allelic specificity by interacting in trans with CWR-2, a predicted transmembrane protein. However, the catalytic activity of CWR-1 is not required for triggering a block in cell fusion. The L2 and LC regions of the CWR-1 PMO domain show high levels of structural variability between different HGs. CWR-1 chimeras containing a LC region from a different HG were sufficient to trigger a cell fusion block, but not quite at wild type levels, suggesting that the complete PMO structure is necessary for allorecognition. Modeling of the transmembrane protein CWR-2 revealed allelic variability in the two major extracellular domains (ED2/ED4). Chimeras of CWR-2 with swapped ED2 or ED4 or ED2/ED4 domains from different cwr-2 haplogroups also altered allelic specificity. SummaryAllorecognition or nonself recognition enables fungi to distinguish genetically different individuals, thereby regulating cooperation to form mycelial networks. This study focused on the cell wall remodeling checkpoint (cwr), where genetic differences in two genes, cwr-1 and cwr-2, triggers allorecognition. Upon cell contact, CWR-1 in one cell functions in trans with CWR-2 in a second cell to confer a block in cell fusion. Chimeric proteins were created and tested to pinpoint domains involved in allelic specificity. For CWR-1, the L2 and LC domains are critical, while for CWR-2, the ED2 and ED4 domains have an important role in regulating cell fusion. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/633681v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@7e150org.highwire.dtl.DTLVardef@1596b00org.highwire.dtl.DTLVardef@1800a37org.highwire.dtl.DTLVardef@11ea8d4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

genetics↗

bioRxiv2022

The C-terminal domain of SEC-10 is fundamental for exocyst function, Spitzenkorper organization and cell morphogenesis in Neurospora crassa.

The exocyst is a conserved multimeric complex that participates in the final steps of the secretion of vesicles. In the filamentous fungus Neurospora crassa, the exocyst is crucial for polar growth, morphology, and the organization of the Spitzenkorper (Spk), the apical body where secretory vesicles accumulate before being delivered to the plasma membrane. In the highly polarized cells of N. crassa, the exocyst subunits SEC-3, SEC-5, SEC-6, SEC-8, and SEC-15 were previously found localized at the plasma membrane of the cells apices, while EXO-70 and EXO-84 occupied the frontal outer layer of the Spk, occupied by vesicles. The localization of SEC-10 had remained so far elusive. In this work, SEC-10 was tagged with the green fluorescent protein (GFP) either at its N- or C-terminus and found localized at the plasma membrane of growing hyphal tips, similar to what was previously observed for some exocyst subunits. While expression of an N-terminally tagged version of SEC-10 at its native locus was fully viable, expression of a C-terminally tagged version at its native locus resulted in severe hyphal growth and polarity defects. Additionally, a sec-10 knockout mutant in a heterokaryotic state (with genetically different nuclei) was viable but showed a strongly aberrant phenotype, confirming that this subunit is essential to maintain hyphal morphogenesis. Transmission electron microscopy analysis revealed the lack of a Spk in the SEC-10-GFP strain, suggesting a critical role of the exocyst in the vesicular organization at the Spk. Mass spectrometry analysis revealed fewer peptides of exocyst subunits interacting with SEC-10-GFP than with GFP-SEC-10, suggesting an essential role of the C-terminus of SEC-10 in exocyst assembly and/or stability. Altogether, our data suggest that an unobstructed C-terminus of SEC-10 is indispensable for the exocyst complex function and that a GFP tag could be blocking important subunit-subunit interactions.

microbiology↗