Biology subjects

McCollum, C.

Publications and source records attributed to McCollum, C..

6 recordsLinked to original sources

bioRxiv2025

Barley C2-Domain Abscisic Acid-Related protein CARa supports susceptibility to Blumeria hordei and localizes to the extrahaustorial membrane

Rho GTPases are key regulators of cellular signalling processes in eukaryotes. In plants, Rho of plants (ROP) proteins function in cell polarization, hormone signalling, and plant immunity or susceptibility to diseases. The barley (Hordeum vulgare) ROP protein RACB is involved in susceptibility towards the biotrophic ascomycete fungus Blumeria hordei (Bh) and supports the accommodation of fungal haustoria. In healthy barley, RACB plays a role in cell development, a function that the pathogen may co-opt for cellular ingrowth of its haustorium into intact epidermal cells of barley. The majority of identified functions of GTP-bound activated RACB appear to be mediated by scaffold proteins. One of them, the ROP Interactive Partner b (RIPb, synonym: Interactor of Constitutive Active ROP b, ICRb) binds to RACB-GTP and co-localizes with RACB at the site of fungal host cell entry. RIPb interacts with RACB-GTP via its C-terminal coiled-coil CC2 domain (RIPbCC2) at the plasma membrane. Here, we show that a barley Calcium-Dependent Phospholipid Binding 2 (C2)-Domain Abscisic Acid-Related protein (CARa) interacts with RIPb and its RIPbCC2 domain in planta. Transient knockdown of CARa renders barley epidermal cells less susceptible to invasion by Bh, whereas overexpression supports fungal haustorium accomodation. Upon fungal attack, CARa is recruited to the site of fungal attack and localizes around the haustorial neck and at the extrahaustorial membrane. These findings further support that Bh profits from host ROP signalling components and CARa. CARa might connect RACB-RIPb-signalling to membrane organization for support of fungal infection structures in barley epidermal cells.

plant biology↗

bioRxiv2021

Structural transitions in the GTP cap visualized by cryo-EM of catalytically inactive microtubules

Microtubules (MTs) are polymers of /{beta}-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes, and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here we use cryo-EM and TIRF microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in -tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice, but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wildtype GDP-MTs. End binding proteins of the EB family have the ability to compact both mutant GTP-lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wildtype MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these first high-resolution structures of truly GTP-bound MTs add mechanistic insight to our understanding of MT dynamic instability. Significance StatementMicrotubules (MTs) are non-equilibrium polymers that switch between states of growth and shrinkage. This property is critical for their function and is a consequence of GTP hydrolysis in the MT. The structure of the stable GTP part of the MT (the GTP cap) has previously been inferred from MTs polymerized with non-hydrolyzable GTP analogs. Here, we report the first high-resolution structures of MTs truly containing GTP, polymerized from mutated, hydrolysis-deficient tubulins. We find that GTP-MTs have an "expanded lattice" and a "closed seam", structural characteristics possibly responsible for stabilizing the GTP cap. These results provide new insight into the structural transitions at growing MT ends, furthering our understanding of the bistable nature of MTs.

biophysics↗

bioRxiv2019

Barley RIC157 is involved in RACB-mediated susceptibility to powdery mildew

Successful obligate pathogens benefit from host cellular processes. For the biotrophic ascomycete fungus Blumeria graminis f.sp. hordei (Bgh) it has been shown that barley RACB, a small monomeric G-protein (ROP, RHO of plants), is required for full susceptibility to fungal penetration. The susceptibility function of RACB probably lies in its role in cell polarisation, which may be co-opted by the pathogen for invasive ingrowth of its haustorium. However, the actual mechanism of how RACB supports the fungal penetration success is little understood. RIC proteins (ROP-Interactive and CRIB-(Cdc42/Rac Interactive Binding) motif-containing) are considered scaffold proteins which can interact directly with ROPs via a conserved CRIB motif. Here we describe a yet uncharacterised RIC protein, RIC157, which can interact directly with RACB in planta. We show that RIC157 undergoes a recruitment from the cytoplasm to the cell periphery in the presence of activated RACB. During fungal infection, RIC157 and activated RACB colocalise at the penetration site, particularly at the haustorial neck. In a RACB-dependent manner, transiently overexpressed RIC157 renders barley epidermal cells more susceptible to fungal penetration. This suggests that RIC157 promotes fungal penetration into barley epidermal cells via its function downstream of RACB.

plant biology↗

bioRxiv2019

ROP INTERACTIVE PARTNER b interacts with the ROP GTPase RACB and supports fungal penetration into barley epidermal cells

RHO of Plants (ROP) G-proteins are key components of cell polarization processes in plant development. The barley (Hordeum vulgare) ROP protein RACB, is a susceptibility factor in the interaction of barley with the barley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh). RACB also drives polar cell development, and this function might be coopted during formation of fungal haustoria in barley epidermal cells. In order to understand RACB signaling during the interaction of barley with Bgh, we searched for potential downstream interactors of RACB. Here, we show that ROP INTERACTIVE PARTNER b (RIPb, synonym: INTERACTOR OF CONSTITUTIVE ACTIVE ROP b; ICRb) directly interacts with RACB in yeast and in planta. Over-expression of RIPb supports susceptibility of barley to Bgh. RIPb further interacts with itself at microtubules. However, the interaction with activated RACB takes place at the plasma membrane. Both, RIPb and RACB are recruited to the site of fungal attack around the neck of developing haustoria suggesting locally enhanced ROP activity. We further assigned different functions to different domains of the RIPb protein. The N-terminal coiled-coil CC1 domain is required for microtubule localization, while the C-terminal coiled-coil CC2 domain is sufficient to interact with RACB and to fulfill a function in susceptibility at the plasma membrane. Hence, RIPb appears to be localized at microtubules and is then recruited by activated RACB for a function at the plasma membrane during formation of the haustorial complex. One Sentence summaryRIPb acts downstream of the powdery mildew susceptibility factor RACB of barley and influences susceptibility

plant biology↗

bioRxiv2019

Barley ROP-INTERACTIVE PARTNER-a organizes into RAC1- and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1-dependent membrane domains

Small ROP (also called RAC) GTPases are key factors in polar cell development and in interaction with the environment. ROP-Interactive Partner (RIP) proteins are predicted scaffold or ROP-effector proteins, which function downstream of activated GTP-loaded ROP proteins in establishing membrane heterogeneity and cellular organization. Grass ROP proteins function in cell polarity, resistance and susceptibility to fungal pathogens but grass RIP proteins are little understood.\n\nWe found that the barley (Hordeum vulgare L.) RIPa protein can interact with barley ROPs in yeast. Fluorescent-tagged RIPa, when co-expressed with the constitutively activated ROP protein CA RAC1, accumulates at the cell periphery or plasma membrane. Additionally, RIPa, locates into membrane domains, which are laterally restricted by microtubules, when co-expressed with RAC1 and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1. Both structural integrity of MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1 and microtubule stability are key to maintenance of RIPa-labeled membrane domains. In this context, RIPa also accumulates at the interface of barley and invading hyphae of the powdery mildew fungus Blumeria graminis f.sp. hordei.\n\nData suggest that barley RIPa interacts with barley ROPs and specifies RAC1 activity-associated membrane domains with potential signaling capacity. Lateral diffusion of this RAC1 signaling capacity is restricted the resulting membrane heterogeneity requires intact microtubules and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1. Focal accumulation of RIPa at sites of fungal attack may indicate locally restricted ROP activity at sites of fungal invasion.

plant biology↗

bioRxiv2018

A Barley Powdery Mildew Fungus Non-Autonomous Retrotransposon Encodes a Peptide that Supports Penetration Success on Barley

Plant immunity is overcome by pathogens by the means of secreted effectors. Host effector targets might be proteins acting in pathogen defense or serve demands of the pathogen. The barley ROP GTPase HvRACB is involved in entry of the powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh) into barley epidermal cells. We found that HvRACB interacts with the ROP-interactive peptide 1 (ROPIP1) that is encoded on the active non-long terminal repeat retroelement Eg-R1 of Bgh. Over-expression of ROPIP1 in barley epidermal cells and host-induced post-transcriptional gene silencing (HIGS) of ROPIP1 suggested that ROPIP1 is involved in virulence of Bgh. Bimolecular fluorescence complementation and co-localization supported that ROPIP1 can interact with activated HvRACB in planta. We show that ROPIP1 is expressed by Bgh on barley and translocated into the cytoplasm of infected barley cells. ROPIP1 is recruited to microtubules upon co-expression of MICROTUBULE ASSOCIATED ROP GTPase ACTIVATING PROTEIN (HvMAGAP1) and can destabilize cortical microtubules. Bgh ROPIP might target HvRACB and manipulate host cell microtubule organization for facilitated host cell entry. Data suggest a possible neo-functionalization of retroelement-derived transcripts for the evolution of a pathogen virulence effector.

plant biology↗