Biology subjects

Kema, G. H. J.

Publications and source records attributed to Kema, G. H. J..

6 recordsLinked to original sources

bioRxiv2026

CRISPR/Cas9-mediated transformation enables functional characterization of the effector Avr4 in the banana pathogen Pseudocercospora fijiensis

Pseudocercospora fijiensis is the causal agent of Black Leaf Streak Disease (BLSD), also known as black Sigatoka, in banana. The disease affects many banana varieties, including the highly susceptible Cavendish banana that dominates global production and the export trade, and several cooking bananas that are a staple food for hundreds of millions of people worldwide. Currently, the disease is controlled using preventative fungicide treatments with up to 70 applications per year in Cavendish plantations, which accounts for approximately 30% of the production costs. Resistant cultivars are required for more sustainable production, but no resistance gene to BLSD has been identified. This is partly due to the poor genetic amenability of P. fijiensis and the lack of methods for functional gene analysis. To address these limitations, we developed a CRISPR/Cas9-mediated transformation system specifically optimized for P. fijiensis. We established a protocol to produce protoplasts, evaluated their capacity to regenerate into new colonies, and assessed antibiotic sensitivity. Subsequently, we confirmed the integration of foreign DNA, including resistance markers, using PEG-mediated transformation. We demonstrated targeted transformation using CRISPR-Cas9 to knockout the polyketide synthase gene PKS10-1, which is responsible for the production of the pigment melanin, and the mitogen-activated protein kinase (MAPK) gene Fus3. Following the successful generation of knockout mutants for these genes, achieving gene targeting efficiencies of respectively 96% and 58%, we subsequently generated knockout mutants of the renowned effector Avr4 in P. fijiensis. The resulting mutants exhibited no reduction in virulence on the susceptible banana cultivar Cavendish. In addition, we used the wild-type isolate and Avr4 knockout strains to test the resistant banana accession Calcutta 4. Contrary to a previous study, we demonstrate that Avr4 does not explain the resistance of Calcutta 4, suggesting that resistance is instead triggered by the recognition of other hitherto unknown effectors. The established CRISPR/Cas9-mediated disruption system is highly efficient and enables routine functional gene characterization, which will help to elucidate genes involved in banana-P. fijiensis interaction, thereby supporting the discovery of resistance genes against BLSD.

molecular biology↗

bioRxiv2025

Transposon activity eliminates a crucial fungal secondary metabolite cluster while preserving pathogenicity

SummaryO_LITransposable elements can significantly influence the genome dynamics of clonal fungal plant pathogens. Tropical Race 4 (TR4) is a clonal lineage within the Fusarium oxysporum species complex that poses a substantial threat to global banana production. However, how transposable elements shape TR4s genome and adaptation remains underexplored. C_LIO_LIWe investigated the activity and impact of FoHeli1, a Helitron transposable element in the TR4 lineage, focusing on a Mozambican TR4 strain (M1) through a combination of genome analyses, metabolite profiling, and infection assays. C_LIO_LIFoHeli1 activity has been very recent within the TR4 lineage and is likely still ongoing. This has resulted in large structural variations in M1, including the loss of the conserved biosynthetic gene cluster required for the production of fusaric acid. We demonstrate that this deletion abolishes fusaric acid production and alters secondary metabolite profiles, but does not affect pathogenicity. C_LIO_LIOur results emphasize the significance of transposable elements, particularly FoHeli1, in reshaping the genetic and metabolic landscape of TR4 and challenge existing assumptions about the role of fusaric acid in pathogenicity. C_LI

microbiology↗

bioRxiv2024

Extensive intrachromosomal duplications in a virulence-associated fungal accessory chromosome

Filamentous fungi have evolved compartmentalized genomes consisting of conserved core regions and dynamic accessory regions, which aid the adaptation to changing environments including the interaction with host organisms. In the Fusarium oxysporum species complex, accessory regions play an important role during infection and it has been reported that these regions undergo extensive duplications, however, it is currently unknown how such duplications shape accessory regions. Moreover, the function of accessory regions apart from encoding virulence effectors is not completely understood. Here we determined the karyotype of F. oxysporum Tropical Race 4 (TR4), which causes the ongoing pandemic of Fusarium wilt of banana (FWB). We show that the single accessory chromosome of TR4 isolate II5 has undergone extensive intrachromosomal duplications, resulting in triplication of the chromosome size compared to other closely related TR4 strains. By obtaining mutant strains that have lost the accessory chromosome, we demonstrate that this chromosome is dispensable for vegetative growth but is required for full virulence on banana. Lastly, we found that the loss of chromosome 12 co-occurs with structural rearrangements of core chromosomes, which are generally co-linear between members of the F. oxysporum species complex. Together, our results provide new insights into the chromosome dynamics of the banana infecting TR4 lineage of the F. oxysporum species complex. SignificanceFusarium oxysporum is a major fungal plant pathogen that causes vascular wilt disease on a wide variety of agronomically important crops. A current epidemic of Fusarium wilt of banana (FWB), caused by tropical race 4 (TR4), poses a major threat to global banana production and threatens food security in tropical and subtropical regions where banana is an important staple crop. Controlling TR4 requires a better understanding of the molecular mechanisms underlying pathogenicity, including the evolution of pathogenicity-related accessory regions. Here we demonstrate that intrachromosomal duplications are a key mechanism of accessory chromosome evolution in the F. oxysporum species complex. We identified a single accessory chromosome and show that TR4 mutants that lost this accessory chromosome display significantly reduced virulence on banana plants. Our results provide insight into the evolution of accessory chromosomes in the F. oxysporum species complex, underscore their importance in pathogenicity, and provide new clues for the development of resistant banana plants.

microbiology↗

bioRxiv2023

Tropical Race 4 and Race 1 strains causing Fusarium wilt of banana infect and survive in Heliconia species and ornamental bananas

Fusarium wilt of banana (FWB), caused by soilborne Fusarium spp., is a major global threat to the cultivation of bananas. In addition to persistent chlamydospores, weeds are a reservoir of the causal agents. However, it remains unclear whether other Zingiberales species, which are grown in the same geographic regions, also can serve as hosts for Fusarium spp. that cause FWB. Greenhouse assays were conducted to investigate whether Fusarium phialophorum (Race 1; pathogenic to Gros Michel banana) and Fusarium odoratissimum (TR4; pathogenic to Cavendish banana) can infect three Heliconia species, two ornamental banana species or Musa textilis (abaca). Heliconia latispatha, Musa balbisiana, and Musa coccinea displayed external symptoms after inoculation with TR4, while inoculation with Race 1 caused symptoms in H. latispatha, H. psittacorum, M. coccinea, and M. velutina. Isolates recovered from distinct organs of all studied plant species were characterized and re-isolated strains caused FWB symptoms in Gros Michel and Cavendish banana plants, and their rhizome discolored area scores were similar to the reference strains. The susceptibility of some ornamental species and the presence of Fusarium strains as asymptomatic endophytes in others, with remaining pathogenicity, call for a revision of the race nomenclature and the current containment protocols for FWB.

plant biology↗

bioRxiv2022

A thousand-genome panel retraces the global spread and climatic adaptation of a major crop pathogen

Human activity impacts the evolutionary trajectories of many species worldwide. Global trade of agricultural goods contributes to the dispersal of pathogens reshaping their genetic makeup and providing opportunities for virulence gains. Understanding how pathogens surmount control strategies and cope with new climates is crucial to predicting the future impact of crop pathogens. Here, we address this by assembling a global thousand-genome panel of Zymoseptoria tritici, a major fungal pathogen of wheat reported in all production areas worldwide. We identify the global invasion routes and ongoing genetic exchange of the pathogen among wheat-growing regions. We find that the global expansion was accompanied by increased activity of transposable elements and weakened genomic defenses. Finally, we find significant standing variation for adaptation to new climates encountered during the global spread. Our work shows how large population genomic panels enable deep insights into the evolutionary trajectory of a major crop pathogen.

genomics↗

bioRxiv2019

Variable Genome Evolution in Fungi After Transposon-Mediated Amplification of a Housekeeping Gene

BackgroundTransposable elements (TEs) can be key drivers of evolution, but the mechanisms and scope of how they impact gene and genome function are largely unknown. Previous analyses revealed that TE-mediated gene amplifications can have variable effects on fungal genomes, from inactivation of function to production of multiple active copies. For example, a DNA methyltransferase gene in the wheat pathogen Zymoseptoria tritici (synonym Mycosphaerella graminicola) was amplified to tens of copies, all of which were inactivated by Repeat-Induced Point mutation (RIP) including the original, resulting in loss of cytosine methylation. In another wheat pathogen, Pyrenophora tritici-repentis, a histone H3 gene was amplified to tens of copies with little evidence of RIP, leading to many potentially active copies. To further test the effects of transposon-aided gene amplifications on genome evolution and architecture, the repetitive fraction of the significantly expanded Pseudocercospora fijiensis genome was analyzed in greater detail. ResultsThese analyses identified a housekeeping gene, histone H3, which was captured and amplified to hundreds of copies by a hAT DNA transposon, all of which were inactivated by RIP, except for the original. In P. fijiensis the original H3 gene probably was not protected from RIP, but most likely was maintained intact due to strong purifying selection. Comparative analyses revealed that a similar event occurred in five additional genomes representing the fungal genera Cercospora, Pseudocercospora and Sphaerulina. ConclusionsThese results indicate that the interplay of TEs and RIP can result in different and unpredictable fates of amplified genes, with variable effects on gene and genome evolution.

genomics↗