Biology subjects

Viljoen, A.

Publications and source records attributed to Viljoen, A..

4 recordsLinked to original sources

bioRxiv2025

Robust design and validation of LAMP assays for in-field detection of three major bacterial vascular diseases of banana

Bacterial diseases of banana are becoming increasingly significant worldwide, resulting in reduced yields and higher disease management costs. The most important bacterial diseases of banana include Moko and banana blood disease (BBD), caused by Ralstonia solanacearum and Ralstonia syzygii subsp. celebesensis, respectively, and banana Xanthomonas wilt (BXW) caused by Xanthomonas vasicola pv. musacearum. Effective surveillance and disease management require point-of-care diagnostics, such as loop-mediated isothermal amplification (LAMP), for on-site operation. In this study, three LAMP assays were developed to specifically detect the bacteria responsible for Moko, BBD and BXW, directly from banana tissues, using a simplified DNA extraction protocol. The BBD - and BXW-LAMP assays demonstrated 100% specificity, yielding negative results for a broad range of non-target bacteria, including closely related species as well as pathogenic and endophytic strains associated with banana, and positive results for all the tested target strains. For Moko disease, a duplex-LAMP assay was developed to detect all strains from the four globally most relevant sequevars: IIB-3, IIB-4, IIA-6, and IIA-24. The duplex-LAMP successfully detected all target strains, except one that was shown to be non-pathogenic to Cavendish bananas. All non-target strains tested negative, with the exception of a delayed signal for one strain belonging to Ralstonia thomasi, not associated with banana environment (hospital strain). These results were supported by an extensive in silico analysis conducted on 9,668 Burkholderiaceae and 7,483 Xanthomonadaceae genomes. Detection limits ranged from 0.1 pg/{micro}l to 1 pg/{micro}l DNA, and from 10 to 10 CFU/ml on banana tissues spiked with calibrated bacterial suspensions, depending on the assay. The LAMP assays prove highly effective for detecting target pathogens in both artificially inoculated banana plants and field samples, offering a promising tool for improving disease management strategies.

molecular biology↗

bioRxiv2025

GRASP: a modular toolkit for synthetic pentatricopeptide repeat RNA-binding proteins

Pentatricopeptide repeat (PPR) proteins are eukaryotic RNA binding proteins with multiple roles in mitochondrial and chloroplast transcript processing. PPR proteins are naturally modular and hold great potential for development into tools for RNA processing or controlling RNA folding or expression. However, construction of synthetic PPR proteins is challenging due to their highly repetitive sequences. Here, we present the GRASP kit for assembly of synthetic PPR proteins. Utilising the S-variant of PPR motifs, we designed a library of 42 plasmids which can be combined to assemble synthetic PPR proteins with 9, 14 or 19 motifs to target any RNA sequence of the same length. The GRASP kit enables rapid design and construction of PPR proteins of any desired specificity and is compatible with the MoClo assembly standard. To demonstrate the capabilities of GRASP, we assembled a synthetic PPR RNA editing protein and variants with altered sequence specificity. We tested the functionality of 31 synthetic PPR protein variants against a set of 46 RNA targets and used RNA sequencing to determine levels of RNA editing. The variations in editing provide a wealth of insights into PPR-RNA interactions. The GRASP kit provides a foundation for further development of synthetic PPR protein technologies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/661641v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@d8c1d2org.highwire.dtl.DTLVardef@9ccba8org.highwire.dtl.DTLVardef@1feadfeorg.highwire.dtl.DTLVardef@150ffb7_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Large pilin subunits provide distinct structural and mechanical properties for the Myxococcus xanthus type IV pilus

Type IV pili (T4P) are ubiquitous bacterial cell surface filaments important for surface motility, adhesion to biotic and abiotic surfaces, DNA uptake, biofilm formation, and virulence. T4P are built from thousands of copies of the major pilin subunit and tipped by a complex composed of minor pilins and in some systems also the PilY1 adhesin. While the major pilins of structurally characterized T4P have lengths of up to 161 residues, the major pilin PilA of Myxococcus xanthus is unusually large with 208 residues. All major pilins have a highly conserved N-terminal domain and a highly variable C-terminal domain, and the additional residues in the M. xanthus PilA are due to a larger C-terminal domain. We solved the structure of the M. xanthus T4P (T4PMx) at a resolution of 3.0 [A] using cryo-electron microscopy (cryo-EM). The T4PMx follows the structural blueprint observed in other T4P with the pilus core comprised of the extensively interacting N-terminal 1-helices while the globular domains decorate the T4P surface. The atomic model of PilA built into this map shows that the large C-terminal domain has much more extensive intersubunit contacts than major pilins in other T4P. As expected from these greater contacts, the bending and axial stiffness of the T4PMx is significantly higher than that of other T4P and supports T4P-dependent motility on surfaces of different stiffnesses. Notably, T4PMx variants with interrupted intersubunit interfaces had decreased bending stiffness and strongly reduced motility on all surfaces. These observations support an evolutionary scenario whereby the large major pilin enables the formation of a rigid T4P that expands the environmental conditions in which the T4P system functions.

microbiology↗

bioRxiv2021

Force-induced changes of PilY1 drive surface sensing by Pseudomonas aeruginosa

During biofilm formation, the opportunistic pathogen Pseudomonas aeruginosa uses its type IV pili (TFP) to sense a surface, eliciting increased second messenger production and regulating target pathways required to adapt to a surface lifestyle. The mechanisms whereby TFP detect surface contact is still poorly understood, although mechanosensing is often invoked with little data supporting this claim. Using a combination of molecular genetics and single cell analysis, with biophysical, biochemical and genomics techniques we show that force-induced changes mediated by the von Willebrand A (vWA) domain-containing, TFP tip-associated protein PilY1 are required for surface sensing. Atomic force microscopy shows that PilY1 can undergo force-induced, sustained conformational changes akin to those observed for mechanosensitive proteins like titin. We show that mutation of a single cysteine residue in the vWA domain results in modestly lower surface adhesion forces, increased nanospring-like properties, as well as reduced c-di-GMP signaling and biofilm formation. Mutating this cysteine has allowed us to genetically separate TFP function in twitching from surface sensing signaling. The conservation of this Cys residue in all P. aeruginosa PA14 strains, and its absence in the ~720 sequenced strains of P. aeruginosa PAO1, could contribute to explaining the observed differences in surface colonization strategies observed for PA14 versus PAO1. ImportanceMost bacteria live on abiotic and biotic surfaces in surface-attached communities known as biofilms. Surface sensing and increased levels of the second messenger molecule c-di-GMP are crucial to the transition from planktonic to biofilm growth. The mechanism(s) underlying TFP-mediated surface detection that triggers this c-di-GMP signaling cascade are unclear. Here, we provide a key insight into this question: we show that the eukaryotic-like, vWA domain of the TFP tip-associated protein PilY1 responds to mechanical force, which in turn drives production of a key second messenger needed to regulate surface behaviors. Our studies highlight a potential mechanism that could account for differing surface colonization strategies.

microbiology↗