Biology subjects

Huguet-Tapia, J. C.

Publications and source records attributed to Huguet-Tapia, J. C..

9 recordsLinked to original sources

bioRxiv2026

SPIN: A Scalable Bioinformatics Pipeline for Screening Pathogenicity Related Host-Pathogen Protein INteractions Using AlphaFold3

Traditional experimental approaches have greatly advanced our understanding of protein-protein interactions (PPIs) that govern host susceptibility or resistance. Nonetheless, the molecular characterization of microbial effectors and their cognate host targets remains challenging in many economically important plant pathosystems. AlphaFold3 (AF3) has transformed structural biology by achieving near-experimental accuracy in protein structure and complex prediction. A bioinformatics pipeline SPIN (Screening Pathogenicity-Related Host/Pathogen Protein INteractions) was developed for large-scale prediction of interactions between the host and pathogen-secreted proteins. SPIN integrates three core modules: pathogen and host protein preprocessing using bioinformatics tools (SignalP6.0, OrthoFinder, CD-HIT), AF3-based interaction modeling, supported by automated input generation and output filtering. However, AF3 training bias toward mammalian proteins necessitated careful evaluation in plant systems. Benchmarking against experimentally validated plant PPIs revealed that derivative metrics emphasizing interfacial geometry and residue-level contact (ipSAE and pDockQ) provide superior discrimination compared to native global confidence measures (pTM and ipTM), particularly for proteins with intrinsically disordered regions. A multi-metric confidence scoring framework combining pLDDT, PAE, ipSAE, and pDockQ, improved prediction reliability by enhancing recall and reduced false positives through robust assessment of structural confidence and interface quality. For proof-of-concept, SPIN was applied to examine the molecular landscape underlying two economically important diseases of citrus (Citrus L.) caused by Candidatus Liberibacter asiaticus and Ca. Phytoplasma citri. Both pathogens are phloem-limited and cause distinct symptoms, citrus greening and witches broom, respectively. AF3-predicted interactome data revealed conserved host colonization strategies alongside disease-specific molecular mechanisms, demonstrating the utility of SPIN for dissecting and supporting mechanistic studies in plant-pathogen interactions.

genomics↗

bioRxiv2026

Genomic Insights into a Multispecies Bacterial Pathogen Complex Driving Bacterial Blotch in White Button Mushrooms.

Bacterial blotch remains a major constraint to global white button mushroom (Agaricus bisporus) industry, yet its etiological complexity has been underestimated. Through a genome resolved, polyphasic approach applied to symptomatic mushrooms collected from United States, we uncovered an unexpectedly diverse complex of Pseudomonas species driving blotch disease. Beyond classical pathogens (P. tolaasii, P. gingeri, P. agarici, and P. "reactans", P. yamanorum, Pseudomonas sp. NC02), our analyses revealed a striking prevalence of P. azotoformans, a species not previously associated with mushroom pathology, alongside P. pergaminensis, P. monsensis, P. tensinigenes, P. simiae, Pseudomonas sp. Irchel 3A7, Pseudomonas sp. REP124 and two putatively novel lineages. Comparative genomics demonstrated pronounced heterogeneity in accessory genome content, with P. azotoformans exhibiting exceptional genomic plasticity indicative of broad ecological adaptability. Secondary metabolite profiling and white line assays further delineated species-specific chemotaxonomic signatures, underscoring the multifactorial nature of virulence. Collectively, this study provides the most comprehensive genomic and phenotypic characterization of blotch-associated Pseudomonas in Northern America, overturning the long-held paradigm of a single dominant pathogen. By establishing that bacterial blotch is multispecies disease complex, our findings redefine its epidemiology and lay the foundation for improved diagnostics strategies in mushroom production systems. The emergence and high prevalence of P. azotoformans underscore the limitations of diagnostic protocols focused exclusively on classical blotch pathogens and highlight the need for broader, genomics informed detection strategies. Collectively, this work offers actionable insights to strengthen production resilience and support the sustainability of white button mushroom cultivation as the worlds most economically important specialty food crop.

microbiology↗

bioRxiv2025

From lab to field: analyses of genome-edited bacterial blight resistant rice

Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the major drivers of yield losses in Africa and Asia. Xoo secretes TAL-effectors (TALe) that induce host SWEET sucrose uniporter by binding to the effector binding element (EBE) of SWEET promoters, likely required for Xoo reproduction and virulence. We had multiplex edited the EBEs of three SWEET genes to prevent TALe binding, producing genome-edited (GEd) rice mega-varieties (IR64, Ciherang-Sub1 for Asia, and Komboka for Africa) that were resistant to a wide spectrum of Xoo strains. Here, we report comprehensive analyses of the GEd lines, including evaluation of agronomic performance in multi-location multi-season experimental field plots under different fertilization regimes, and tests for the presence/absence of foreign DNA/transgene in the offspring of GEd lines (IR64-BC1T6, Ciherang-Sub1-BC1T5, Komboka-T3). Various strategies were evaluated, including herbicide tolerance, PCR, DNA gel blotting, whole genome sequencing (WGS), and specific tests stipulated by country-specific biosafety guidelines. Different WGS technologies were evaluated and also used to identify heritability of the edits, single nucleotide polymorphisms (SNPs), and insertions/deletions (indels) that might have resulted from somaclonal variation and potential GE-induced off-target mutations. Complete genome reference sequences for the parental lines IR64, Ciherang-Sub1, and Komboka are provided. In the field experiments, the GEd lines did not show performance defects. Together, the results indicate that select GE lines do not contain foreign DNA or transgene fragments and fulfill the requirements for treatment equivalent to classical breeding lines in countries such as India and Kenya.

plant biology↗

bioRxiv2025

Interactions Between Native Soil Microbiome and a Synthetic Microbial Community Reveals Bacteria with Persistent Traits

Synthetic microbial communities (SynComs) are curated microbial groups designed to enhance plant growth or disease resistance by augmenting soil microbiomes. Attaining SynCom stability in the presence of native soil communities remains a key challenge. This study investigated the survival, persistence, and chemical interactions of a SynCom with a native soil microbial community using a transwell system that spatially constrains bacteria while permitting chemical interactions. The SynCom, composed of six compatible Pseudomonas species identified through whole-genome sequencing, was analyzed for antagonistic interactions with native microbes over time and assessed using biomass and viability measurements. Over time, the SynCom exhibitedreduced growth in the presence of native soil microbes compared to the SynCom not exposed to the native microbes. Flow cytometry analysis showed an 81% reduction of live cells for the persistent strain in the presence of native microbes and a 78% and 99% increase in dead and unstained cells, respectively. Compared to a non-persistent strain, one persistent SynCom strain showed lower metabolic utilization across five key compound classes: polymers, carboxylic acids, amino acids, amines, and phenols when exposed to the native soil microbes. These findings underscore the importance of understanding complex SynCom-environment interactions to enhance SynCom stability and optimize in situ applications. ImportanceSynthetic Microbial Communities, or SynComs, are an emerging technology that can potentially augment plant health. Still, their application in situ depends on deciphering the complex interactions between SynCom microbes and native microbial communities. This study provides insight into several Pseudomonas strains displaying persistent characteristics, which makes these bacteria promising candidates for SynCom stability in implanted environments. Understanding the persistent traits of these bacteria is a vital advancement in SynCom technology, and an important next step toward implementing SynComs in agricultural systems.

microbiology↗

bioRxiv2023

Genome analysis and hyphal movement characterization of the hitchhiker endohyphal Enterobacter sp. from Rhizoctonia solani

Bacterial-fungal interactions are pervasive in the rhizosphere. While an increasing number of endohyphal bacteria (EHB) have been identified, little is known about their ecology and impact on the associated fungal hosts and the surrounding environment. In this study, we characterized the genome of an Enterobacter sp. (En-Cren) isolated from the generalist fungal pathogen Rhizoctonia solani. Overall, the En-Cren genome size was typical for members of the genus and was capable of free-living growth. The genome was 4.6 MB in size, and no plasmids were detected. Several prophage regions and genomic islands were identified that harbor unique genes in comparison with phylogenetically closely related Enterobacter spp. Type VI secretion system and cyanate assimilation genes were identified from the bacterium, while common heavy metal resistance genes were absent. En-Cren contains the key genes for indole-3-acetic acid (IAA) and phenylacetic acid (PAA) biosynthesis, and produces IAA and PAA in vitro, which may impact the ecology or pathogenicity of the fungal pathogen in vivo. En-Cren was observed to move along hyphae of R. solani and on other basidiomycetes and ascomycetes in culture. The bacterial flagellum is essential for hyphal movement, while other pathways and genes may also be involved. ImportanceThe genome characterization and comparative genomics analysis of En-Cren provided the foundation and resources for a better understanding of the ecology and evolution of this EHB in the rhizosphere. The ability to produce IAA and PAA may provide new angles to study the impact of phytohormones during the plant-pathogen interactions. The hitchhiking behavior of the bacterium on a diverse group of fungi, while inhibiting the growth of some others, revealed new areas of bacterial-fungal signaling and interaction yet to be explored.

microbiology↗

bioRxiv2022

Genome-wide identification, characterization, and comparative analysis of NLR resistance genes in Coffea spp.

The largest family of disease resistance genes in plants are nucleotide-binding site leucine-rich repeat genes (NLRs). The products of these genes are responsible for recognizing avirulence proteins (Avr) of phytopathogens and triggering specific defense responses. Identifying NLRs in plant genomes with standard gene annotation software is challenging due to their multidomain nature, sequence diversity, and clustered genomic distribution. We present the results of a genome-wide scan and comparative analysis of NLR loci in three coffee species (Coffea canephora, Coffea eugenioides and their interspecific hybrid Coffea arabica). A total of 1311 non-redundant NLR loci were identified in C. arabica, 927 in C. canephora, and 1079 in C. eugenioides, of which 809, 562, and 695 are complete loci, respectively. The NLR-annotator tool used in this study showed extremely high sensitivities and specificities (over 99%) and increased the detection of putative NLRs in the reference coffee genomes. The NLRs loci in coffee are distributed among all chromosomes and are organized mostly in clusters. The C. arabica genome presented a smaller number of NLR loci when compared to the sum of the parental genomes (C. canephora, and C. eugenioides). There are orthologous NLRs (orthogroups) shared between coffee, tomato, potato, and reference NLRs and those that are shared only among coffee species, which provides clues about the functionality and evolutionary history of these orthogroups. Phylogenetic analysis demonstrated orthologous NLRs shared between C. arabica and the parental genomes and those that were possibly lost. The NLR family members in coffee are subdivided into two main groups: TIR-NLR (TNL) and non-TNL. The non-TNLs seem to represent a repertoire of resistance genes that are important in coffee. These results will support functional studies and contribute to a more precise use of these genes for breeding disease-resistant coffee cultivars.

plant biology↗

bioRxiv2021

Phyllosphere-inhabiting endophytic bacteria feature a stationary phase-like lifestyle

Plants are in constant association with a variety of microbes, and although much is known about how symbiotic and pathogenic microbes interact with plants, less is known about the population dynamics, adaptive traits, and transcriptional features of endophytic commensal microbes that live inside leaves. In this study, we evaluated the long-term population and transcriptional dynamics of two bacterial microbiota endophytes and compared them to those of a commensal-simulating non-pathogenic mutant of the bacterial pathogen Pseudomonas syringae. We found that population densities of all three endophytic phyllosphere bacteria remained static over a long period of time, which was caused by a continual equilibrium between bacterial multiplication and death, as evidenced by treatment of plants with antibiotics that only targeted dividing bacteria or by in planta visualization of bacteria carrying a fluorescent division reporter. Population stasis could not be explained by a lack of resources, as Arabidopsis leaves could support population densities up to 100 times higher than the normal microbiota populations, nor was population stasis reversed by significantly quenching PAMP-triggered immunity. Long-term temporal in planta transcriptomic analysis of these three bacterial endophytes revealed up-regulation of protein translation, the generation of energy, and the response to stress, and interestingly, for the microbiota strains, the longer the bacteria remained inside plants, the greater the up-regulation of some of these processes. Further transcriptomic analysis of in planta populations of commensal-simulating Pseudomonas syringae revealed a remarkable resemblance to those of in vitro bacteria in stationary phase, a metabolically active physiological state in which the production of secondary metabolites and stress responses are induced. This study provides novel insight into how endophytic bacteria survive and thrive inside plant leaves, and reshapes our current understanding of what it means to be part of the endophytic microbiota in the phyllosphere.

microbiology↗

bioRxiv2019

An efficient method to clone TAL effector genes from Xanthomonas oryzae using Gibson assembly

TALes (Transcription Activator-Like effectors) represent the largest family of type III effectors among pathogenic bacteria and play a critical role in the process of infection. Strains of Xanthomonas oryzae pv. oryzae (Xoo) and some strains of other Xanthomonas pathogens contain large numbers of TALe genes. Previous techniques to clone individual or a complement of TALe genes through conventional strategies are inefficient and time-consuming due to multiple genes (up to 29 copies) in a given genome and technically challenging due to the repetitive sequences (up to 33 nearly identical 102-nucleotide repeats) of individual TALe genes. Thus, only a limited number of TALe genes have been molecularly cloned and characterized, and the functions of most TALe genes remain unknown. Here, we present an easy and efficient cloning technique to clone TALe genes selectively through in vitro homologous recombination and single strand annealing and demonstrate the feasibility of this approach with four different Xoo strains. Based on the Gibson assembly strategy, two complementary vectors with scaffolds that can preferentially capture all TALe genes from a pool of genomic fragments were designed. Both vector systems enabled cloning of a full complement of TALe genes from each of four Xoo strains and functional analysis of individual TALes in rice in approximately one month compared to three months by previously used methods. The results demonstrate a robust tool to advance TALe biology and a potential for broad usage of this approach to clone multiple copies of highly competitive DNA elements in any genome of interest.

microbiology↗

bioRxiv2019

Transcriptome profile of Carrizo citrange roots in response to Phytophthora parasitica infection

Phytophthora parasitica is one of the most widespread Phytophthora species, which is known to cause root rot, foot rot/gummosis and brown rot of fruits in citrus. In this study, we have analyzed the transcriptome of a commonly used citrus rootstock Carrizo citrange in response to P. parasitica infection using the RNA-seq technology. In total, we have identified 6692 differentially expressed transcripts (DETs) among P. parasitica-inoculated and mock-treated roots. Of these, 3960 genes were differentially expressed at 24 hours post inoculation and 5521 genes were differentially expressed at 48 hours post inoculation. Gene ontology analysis of DETs suggested substantial transcriptional reprogramming of diverse cellular processes particularly the biotic stress response pathways in Carrizo citrange roots. Many R genes, transcription factors, and several other genes putatively involved in plant immunity were differentially modulated in citrus roots in response to P. parasitica infection. Analysis reported here lays out a strong foundation for future studies aimed at improving resistance of citrus rootstocks to P. parasitica.

genomics↗