Insight into the molecular requirements for pathogenicity of Fusarium oxysporum f. sp. lycopersici through large-scale insertional mutagenesis

An insertional mutagenesis screen identifies pathogenicity-related genes in the plant fungal pathogen Fusarium oxysporum

Rapid evolution in plant-microbe interactions - a molecular genomics perspective

Rapid (co-)evolution at multiple timescales is a hallmark of plant-microbe interactions. The mechanistic basis for the rapid evolution largely rests on the features of the genomes of the interacting partners involved. Here, we review recent insights into genomic characteristics and mechanisms that enable rapid evolution of both plants and phytopathogens. These comprise fresh insights in allelic series of matching pairs of resistance and avirulence genes, the generation of novel pathogen effectors, the recently recognised small RNA warfare, and genomic aspects of secondary metabolite biosynthesis. In addition, we discuss the putative contributions of permissive host environments, transcriptional plasticity and the role of ploidy on the interactions. We conclude that the means underlying the rapid evolution of plant-microbe interactions are multifaceted and depend on the particular nature of each interaction

Insights into Adaptations to a Near- Obligate Nematode Endoparasitic Lifestyle from the Finished Genome of Drechmeria coniospora

Nematophagous fungi employ three distinct predatory strategies: nematode trapping, parasitism of females and eggs, and endoparasitism. While endoparasites play key roles in controlling nematode populations in nature, their application for integrated pest management is hindered by the limited understanding of their biology. We present a comparative analysis of a high quality finished genome assembly of Drechmeria coniospora, a model endoparasitic nematophagous fungus, integrated with a transcriptomic study. Adaptation of D. coniospora to its almost completely obligate endoparasitic lifestyle led to the simplification of many orthologous gene families involved in the saprophytic trophic mode, while maintaining orthologs of most known fungal pathogen-host interaction proteins, stress response circuits and putative effectors of the small secreted protein type. The need to adhere to and penetrate the host cuticle led to a selective radiation of surface proteins and hydrolytic enzymes. Although the endoparasite has a simplified secondary metabolome, it produces a novel peptaibiotic family that shows antibacterial, antifungal and nematicidal activities. Our analyses emphasize the basic malleability of the D. coniospora genome: loss of genes advantageous for the saprophytic lifestyle; modulation of elements that its cohort species utilize for entomopathogenesis; and expansion of protein families necessary for the nematode endoparasitic lifestyle

Tomato R-gene-mediated resistance against Fusarium wilt originates in roots and extends to shoots via xylem to limit pathogen colonization

Vascular wilt disease, caused by the soil-borne fungus Fusarium oxysporum (Fo), poses a threat to many crop species. Four different tomato resistance (R) genes (I-1, I-2, I-3, and I-7) have been identified to confer protection against Fo f.sp. lycopersici (Fol). These I genes are root-expressed and mount an immune response upon perception of the invading fungus. Despite immune activation, Fol is still able to colonize the xylem vessels of resistant tomato lines. Yet, the fungus remains localized in the vessels and does not colonize adjacent tissues or cause disease. The molecular mechanism constraining Fol in the vascular system of the stem remains unclear. We here demonstrate that an I-2-resistant rootstock protects a susceptible scion from Fusarium wilt, notwithstanding fungal colonization of the susceptible scion. Proteomic analyses revealed the presence of fungal effectors in the xylem sap of infected plants, showing that the lack of fungal pathogenicity is not due to its inability to express its virulence genes. To identify mobile root-derived proteins, potentially involved in controlling fungal proliferation, comparative xylem sap proteomics was performed. We identified distinct pathogenesis-related (PR) protein profiles in xylem sap from Fol-inoculated I-1, I-2, I-3, and I-7 resistant lines. Despite structural diversity, all four immune receptors trigger the accumulation of a common set of four PR proteins: PR-5x, PR-P2, and two glucan endo-1,3-β-D-glucosidases. This research provides insights into Fusarium resistance mechanisms and identifies a core set of proteins whose abundance correlates with defense against Fusarium wilt

Divergent ECC1 effector homologs modulate host-specific virulence in cucurbit-infecting Fusarium oxysporum

Fusarium oxysporum (Fo) is a soil-borne fungal pathogen that causes wilt disease in over one hundred plant species, with host-specific strains classified into formae speciales (ff. spp.). For example, Fo f. sp. melonis (Fom) only causes disease in melon while Fo f. sp. radicis-cucumerinum (Forc) can infect multiple cucurbit species. The virulence factors underlying host specificity in these cucurbit-infecting formae speciales have largely remained elusive, limiting our understanding of Fo-host interactions. A previous study identified Effector for Cucurbit Compatibility 1a (ECC1a), an avirulence protein from Fom that restricts cucumber infection when introduced into Forc. Here, we show that ECC1a is part of a previously unrecognized effector gene family, ECC1, abundantly present in strains that infect one or more cucurbit species. However, the role of this family in host compatibility is still poorly understood. Using gene knockout- and replacement strategies, we show that the ECC1 gene family contributes to virulence of both Forc and Fom on cucumber and melon. Specifically, ECC1a contributes to Fom virulence on melon and Forc virulence on cucumber. ECC1b appears to be primarily involved in Fom virulence on melon.Expression profiling reveals a potential role of ECC1 during early stages of infection, suggesting involvement in initial host colonization. Together, these findings highlight the host- and forma specialis-specific functions of ECC1 homologs in Fo pathogenicity

Evaluation of Some Specific Primer Sets Development for Detecting Fusarium oxysporum f. sp. cubense Tropic Race 4 (Foc TR4) Originating from Indonesia

Fusarium oxysporum f. sp. cubense tropic race 4 (Foc TR4) strain which belong to Vegetative Compatibility Group (VCG) 01213 is the most devastating disease in global banana production. Validation of specific primer sets using the positive control (Foc TR4). In total, 50 isolates of Foc are collected from several banana production regions in Indonesia represent the group of VCG, races, genotype, cultivars, which are confirmed as Foc based on the tested using FocEf3 primer set, except Cjr-2 and Lmp-4 isolates. Foc-1/Foc-2 could amplify 34 Foc isolates included in Foc race 4. Three specific primer sets i.e. TR4-F/TR4-R, Six-1c, and TR4-F2/TR4-R1 are used to classify Foc isolates into Foc tropic race 4. TR4-F/TR4-R is known have the highest specificity as it could amplify 35 Foc isolates including positive controls (Foc TR4) compared to the other primer sets (Six-1c and TR4-F2/TR4-R1). This research indicates that there are a large number of diversity strains found in Foc isolates to be studied for further research. Race 4 of Foc (STR4 or TR4) is known to be widespread in several regions in Indonesia. Therefore, specific primer set development needs to be done to detect Foc TR4 and the most damaging strains on Foc TR4 based on molecular data. Intisari Fusarium oxysporum f. sp. cubense ras 4 tropika (Foc TR4) yang termasuk ke dalam kelompok VCG 01213 merupakan patogen yang paling merusak dalam produksi tanaman pisang secara global. Validasi primer spesifik berbasis PCR menggunakan kontrol positif (Foc TR4). Total, 50 isolat Foc dikoleksi dari  beberapa daerah produksi pisang di Indonesia mewakili VCG, ras, genotipe dan kultivar yang dikonfirmasi sebagai isolat Foc berdasarkan pengujian menggunakan primer FocEf3, kecuali isolat Cjr-2 dan Lmp-4. Foc-1/Foc-2 dapat mengamplifikasi 34 isolat Foc yang termasuk ke dalam Foc ras 4. Selanjutnya tiga pasang primer spesifik yaitu TR4-F/TR4-R, Six-1c, dan TR4-F2/TR4-R1 digunakan untuk mengelompokkan isolat-isolat tersebut ke dalam isolat Foc ras 4 tropika. TR4-F/TR4-R diketahui memiliki spesifitas tertinggi karena dapat mengamplifikasi sebanyak 35 isolat Foc termasuk kontrol positif (Foc TR4) dibandingkan dengan primer lainnya (Six-1c dan TR4-F2/TR4-R1). Penelitian ini menunjukkan bahwa terdapat sejumlah besar keragaman strain yang terlihat pada isolat-isolat Foc tersebut untuk dapat dipelajari lebih lanjut. Ras 4 dari Foc (STR4 atau TR4) diketahui tersebar luas pada beberapa daerah di Indonesia. Oleh karena itu, perlu dilakukan pengembangan primer spesifik untuk mendeteksi Foc TR4 dan strain yang paling merusak pada Foc TR4 berdasarkan data molekuler

The tomato xylem sap protein XSP10 is required for full susceptibility to Fusarium wilt disease

XSP10 is an abundant 10 kDa protein found in the xylem sap of tomato. The protein displays structural similarity to plant lipid transfer proteins (LTPs). LTPs are involved in various physiological processes, including disease resistance, and some are able to bind and transfer diverse lipid molecules. XSP10 abundance in xylem sap declines upon infection with Fusarium oxysporum f. sp. lycopersici (Fol), implying involvement of XSP10 in the plant–pathogen interaction. Here, the biochemical characterization of XSP10 with respect to fatty acid-binding properties is reported; a weak but significant binding to saturated fatty acids was found. Furthermore, XSP10-silenced tomato plants were engineered and it was found that these plants exhibited reduced disease symptom development upon infection with a virulent strain of Fol. Interestingly, the reduced symptoms observed did not correlate with an altered expression profile for known reporter genes of plant defence (PR-1 and WIPI). This work demonstrates that XSP10 has lipid-binding properties and is required for full susceptibility of tomato to Fusarium wilt

The Arabidopsis leucine-rich repeat receptor kinase MIK2/LRR-KISS connects cell wall integrity sensing, root growth and response to abiotic and biotic stresses

Plants actively perceive and respond to perturbations in their cell walls which arise during growth, biotic and abiotic stresses. However, few components involved in plant cell wall integrity sensing have been described to date. Using a reverse-genetic approach, we identified the Arabidopsis thaliana leucine-rich repeat receptor kinase MIK2 as an important regulator of cell wall damage responses triggered upon cellulose biosynthesis inhibition. Indeed, loss-of-function mik2 alleles are strongly affected in immune marker gene expression, jasmonic acid production and lignin deposition. MIK2 has both overlapping and distinct functions with THE1, a malectin-like receptor kinase previously proposed as cell wall integrity sensor. In addition, mik2 mutant plants exhibit enhanced leftward root skewing when grown on vertical plates. Notably, natural variation in MIK2 (also named LRR-KISS) has been correlated recently to mild salt stress tolerance, which we could confirm using our insertional alleles. Strikingly, both the increased root skewing and salt stress sensitivity phenotypes observed in the mik2 mutant are dependent on THE1. Finally, we found that MIK2 is required for resistance to the fungal root pathogen Fusarium oxysporum. Together, our data identify MIK2 as a novel component in cell wall integrity sensing and suggest that MIK2 is a nexus linking cell wall integrity sensing to growth and environmental cues

Xylem Sap Proteomics Reveals Distinct Differences Between R Gene- and Endophyte-Mediated Resistance Against Fusarium Wilt Disease in Tomato

Resistance (R) genes and endophytic organisms can both protect plants against pathogens. Although the outcome of both processes is the same, little is known about the commonalities and differences between both immune responses. Here we set out to phenotypically characterize both responses in the tomato-Fusarium pathosystem, and to identify markers to distinguish these responses at the molecular level. As endophyte Fusarium oxysporum (Fo) strain Fo47 was employed, which confers protection against various pathogens, including the vascular wilt fungus F. oxysporum f.sp. lycopersici (Fol). As R-gene conferring Fol resistance, the I-2 gene of tomato (Solanum lycopersicum) was used. Fol colonizes the xylem vessels of susceptible and I-2 resistant tomato plants, but only causes disease in the former. Fol was found to colonize the vasculature of endophyte-colonized plants, and could be isolated from stems of non-diseased plants co-inoculated with Fo47 and Fol. Because the xylem vessels form the main interface between plant and pathogen, the xylem sap proteomes during R gene- and Endophyte-Mediated Resistance (RMR and EMR) were compared using label-free quantitative nLC-MS/MS. Surprisingly, both proteomes were remarkably similar to the mock, revealing only one or two differentially accumulated proteins in the respective resistant interactions. Whereas in I-2 plants the accumulation of the pathogenesis-related protein PR-5x was strongly induced by Fol, the endophyte triggered induction of both NP24, another PR-5 isoform, and of a β-glucanase in the presence of Fol. Notably, over 54% of the identified xylem sap proteins have a predicted intracellular localization, which implies that these might be present in exosomes. In conclusion, whereas both resistance mechanisms permit the pathogen to colonize the vasculature, this does not result in disease and this resistance coincides with specific induction of two distinct PR-5 isoforms and a β-glucanase