Dispensable chromosomes in Fusarium oxysporum f. sp. lycopersici.

The genomes of many filamentous fungi consist of a 'core' part containing conserved genes essential for normal development as well as conditionally dispensable (CD) or lineage-specific (LS) chromosomes. In the plant-pathogenic fungus Fusarium oxysporum f. sp. lycopersici, one LS chromosome harbours effector genes that contribute to pathogenicity. We employed flow cytometry to select for events of spontaneous (partial) loss of either the two smallest LS chromosomes or two different core chromosomes. We determined the rate of spontaneous loss of the 'effector' LS chromosome in vitro at around 1 in 35 000 spores. In addition, a viable strain was obtained lacking chromosome 12, which is considered to be a part of the core genome. We also isolated strains carrying approximately 1-Mb deletions in the LS chromosomes and in the dispensable core chromosome. The large core chromosome 1 was never observed to sustain deletions over 200 kb. Whole-genome sequencing revealed that some of the sites at which the deletions occurred were the same in several independent strains obtained for the two chromosomes tested, indicating the existence of deletion hotspots. For the core chromosome, this deletion hotspot was the site of insertion of the marker used to select for loss events. Loss of the core chromosome did not affect pathogenicity, whereas loss of the effector chromosome led to a complete loss of pathogenicity.

Exchange of core chromosomes and horizontal transfer of lineage-specific chromosomes in Fusarium oxysporum.

Horizontal transfer of supernumerary or lineage-specific (LS) chromosomes has been described in a number of plant pathogenic filamentous fungi. So far it was not known whether transfer is restricted to chromosomes of certain size or properties, or whether 'core' chromosomes can also undergo horizontal transfer. We combined a directed and a non-biased approach to determine whether such restrictions exist. Selection genes were integrated into the genome of a strain of Fusarium oxysporum pathogenic on tomato, either targeted to specific chromosomes by homologous recombination or integrated randomly into the genome. By testing these strains for transfer of the marker to another strain we could confirm transfer of a previously described mobile pathogenicity chromosome. Surprisingly, we also identified strains in which (parts of) core chromosomes were transferred. Whole genome sequencing revealed that this was accompanied by the loss of the homologous region from the recipient strain. Remarkably, transfer of the mobile pathogenicity chromosome always accompanied this exchange of core chromosomes.

Fluorescence Assisted Selection of Transformants (FAST): Using flow cytometry to select fungal transformants.

The availability of drug resistance markers for fungal transformation is often a limiting factor in both fungal genetics research and industrial applications. We describe a new technique using flow cytometry to select fungal transformants using well-known fluorescent proteins as markers for transformation. This new technique, Fluorescence-Assisted Selection of Transformants (FAST), was used for a transformation of Fusarium oxysporum with GFP as a marker targeted at a specific site on chromosome 14. The resulting strain was then transformed again with a gene replacement construct containing both RFP and a gene for drug resistance as markers. By directly comparing FAST with drug resistance selection we show that both methods yield comparable numbers of gene deletion mutants.

Root defense analysis against Fusarium oxysporum reveals new regulators to confer resistance.

Fusarium oxysporum is a root-infecting fungal pathogen that causes wilt disease on a broad range of plant species, including Arabidopsis thaliana. Investigation of the defense response against this pathogen had primarily been conducted using leaf tissue and little was known about the root defense response. In this study, we profiled the expression of root genes after infection with F. oxysporum by microarray analysis. In contrast to the leaf response, root tissue did not show a strong induction of defense-associated gene expression and instead showed a greater proportion of repressed genes. Screening insertion mutants from differentially expressed genes in the microarray uncovered a role for the transcription factor ETHYLENE RESPONSE FACTOR72 (ERF72) in susceptibility to F. oxysporum. Due to the role of ERF72 in suppressing programmed cell death and detoxifying reactive oxygen species (ROS), we examined the pub22/pub23/pub24 U-box type E3 ubiquitin ligase triple mutant which is known to possess enhanced ROS production in response to pathogen challenge. We found that the pub22/23/24 mutant is more resistant to F. oxysporum infection, suggesting that a heightened innate immune response provides protection against F. oxysporum. We conclude that root-mediated defenses against soil-borne pathogens can be provided at multiple levels.

Rewiring of the Jasmonate Signaling Pathway in Arabidopsis during Insect Herbivory.

Plant defenses against insect herbivores and necrotrophic pathogens are differentially regulated by different branches of the jasmonic acid (JA) signaling pathway. In Arabidopsis, the basic helix-loop-helix leucine zipper transcription factor (TF) MYC2 and the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) domain TF ORA59 antagonistically control these distinct branches of the JA pathway. Feeding by larvae of the specialist insect herbivore Pieris rapae activated MYC2 transcription and stimulated expression of the MYC2-branch marker gene VSP2, while it suppressed transcription of ORA59 and the ERF-branch marker gene PDF1.2. Mutant jin1 and jar1-1 plants, which are impaired in the MYC2-branch of the JA pathway, displayed a strongly enhanced expression of both ORA59 and PDF1.2 upon herbivory, indicating that in wild-type plants the MYC2-branch is prioritized over the ERF-branch during insect feeding. Weight gain of P. rapae larvae in a no-choice setup was not significantly affected, but in a two-choice setup the larvae consistently preferred jin1 and jar1-1 plants, in which the ERF-branch was activated, over wild-type Col-0 plants, in which the MYC2-branch was induced. In MYC2- and ORA59-impaired jin1-1/RNAi-ORA59 plants this preference was lost, while in ORA59-overexpressing 35S:ORA59 plants it was gained, suggesting that the herbivores were stimulated to feed from plants that expressed the ERF-branch rather than that they were deterred by plants that expressed the MYC2-branch. The feeding preference of the P. rapae larvae could not be linked to changes in glucosinolate levels. Interestingly, application of larval oral secretion into wounded leaf tissue stimulated the ERF-branch of the JA pathway, suggesting that compounds in the oral secretion have the potential to manipulate the plant response toward the caterpillar-preferred ERF-regulated branch of the JA response. Our results suggest that by activating the MYC2-branch of the JA pathway, plants prevent stimulation of the ERF-branch by the herbivore, thereby becoming less attractive to the attacker.