Involvement of Botrytis cinerea Small GTPases BcRAS1 and BcRAC in Differentiation, Virulence, and the Cell Cycle

Abstract

bSmall GTPases of the Ras superfamily are highly conserved proteins that are involved in various cellular processes, in particular morphogenesis, differentiation, and polar growth. Here we report on the analysis of RAS1 and RAC homologues from the gray mold fungus Botrytis cinerea. We show that these small GTPases are individually necessary for polar growth, reproduction, and pathogenicity, required for cell cycle progression through mitosis (BcRAC), and may lie upstream of the stress-related mitogenactivated protein kinase (MAPK) signaling pathway. bcras1 and bcrac deletion strains had reduced growth rates, and their hyphae were hyperbranched and deformed. In addition, both strains were vegetatively sterile and nonpathogenic. A strain expressing a constitutively active (CA) allele of the BcRAC protein had partially similar but milder phenotypes. Similar to the deletion strains, the CA-BcRAC strain did not produce any conidia and had swollen hyphae. In contrast to the two deletion strains, however, the growth rate of the CA-BcRAC strain was normal, and it caused delayed but well-developed disease symptoms. Microscopic examination revealed an increased number of nuclei and disturbance of actin localization in the CA-BcRAC strain. Further work with cell cycle-and RAC-specific inhibitory compounds associated the BcRAC protein with progression of the cell cycle through mitosis, possibly via an effect on microtubules. Together, these results show that the multinucleate phenotype of the CA-BcRAC strain could result from at least two defects: disruption of polar growth through disturbed actin localization and uncontrolled nuclear division due to constitutive activity of BcRAC.

Function of small GTPase Rho3 in regulating growth, conidiation and virulence of Botrytis cinerea

“…By analyzing the localization of green fluorescent protein-tagged Rho4, they proved that the Rho4 had different roles in the regulation of septation during conidiation versus vegetative septation in filamentous fungi N. crassa. Recent reports showed that the Rac and Cdc42, two GTPase proteins of B. cinerea, were important for cell cycle by acting on the nuclear division (Kokkelink et al, 2011;Minz-Dub et al, 2013). The role of Rho3 in regulation of cell cycle is worthy to further study.…”

“…The Rho superfamily contains three kinds of GTPase named Rac, Cdc42, and Rho, which regulate a wide spectrum of cellular functions (Kwon et al, 2011). Minz-Dub et al (2013) found that RAC protein was involved in the growth, differentiation and the virulence by regulating the actin localization and nuclear division in Botrytis cinerea. Rho3, one monomeric GTPase of the Rho superfamily, is found only in fungi.…”

Small-GTPase-Associated Signaling by the Guanine Nucleotide Exchange Factors CpDock180 and CpCdc24, the GTPase Effector CpSte20, and the Scaffold Protein CpBem1 in Claviceps purpurea

“…In fungi, highly conserved members of the Rho-GTPase family, Cdc42 and Rac1, are crucial for establishing and maintaining polarity during filament formation and budding (7)(8)(9)(10). The importance of Rac homologues in comparison with Cdc42 homologues seems to increase with the developmental complexity of the fungal species (10)(11)(12)(13)(14). In C. purpurea, the two GTPases show overlapping and differing functions.…”

Orchestration of morphogenesis in filamentous fungi: conserved roles for Ras signaling networks

“…In the gray mold, B. cinerea , bcras2 deletion leads to slowed germination and greatly reduced hyphal growth (Minz Dub et al, 2013). In spite of this, the Δ bcras2 strain is able to infect bean leaves, albeit with a delayed disease presentation.…”

“…It is important to note that the Ras1-like and Ras2-like homologs of filamentous fungi typically encode these differing localization motifs (Figure 2), as this dichotomy suggests the potential for differential sub-cellular localizations and, therefore, potentially unique signaling partners under physiological conditions. In support of this possibility, Ras2-like homologs have been deleted in many filamentous fungi whereas deletion of Ras1-like homologs has only been achieved for A. fumigatus , T. reesei and B. cinerea (Fortwendel et al, 2008; Minz Dub et al, 2013; Zhang et al, 2012a). The Ras1- and Ras2-like homolog paradigm is in contrast to the S. cerevisiae model, where Ras1p and Ras2p are highly homologous and have identical prenylation and palmitoylation domains, or S. pombe where only one Ras homolog exists (Weeks and Spiegelman, 2003).…”