The protein modifier SUMO is critical for integrity of the Arabidopsis shoot apex at warm ambient temperatures.

SUMO is a protein modification whose conjugate levels peak during acute heat stress. We find that SUMO is also critical for plant longevity when Arabidopsis experiences a prolonged non-damaging period of only 28 degrees Celsius. Remarkably, this thermo-lethality at 28 degrees was not seen with any other mutant of the SUMO pathway tested. Autoimmunity due to low SUMO1/2 expression levels was not causal for this thermo-lethality. The role of SUMO for thermo-resilience was also distinct from its requirement for thermomorphogenesis - a growth response triggered by the same warm temperature, as only the latter response was dependent on the SUMO ligase SIZ1 as well. Thermo-resilience at 28 degrees Celsius and (acquired) thermotolerance (a response that allows plants to recover and acclimate to brief extreme temperatures) both depend on the HEAT SHOCK TRANSCRIPTION FACTOR A1 (HSFA1). Acquired thermotolerance was, however, normal in the sumo1/2 knockdown mutant. Thus, SUMO-dependent thermo-resilience is potentially controlled in a different way than the protein damage pathway that underpins thermotolerance. Close inspection of shoot apices revealed that the cell patterning and tissue integrity of the shoot apex of the SUMO1/2 knockdown mutant was lost at 28, but not 22 degrees Celsius. We thus describe a novel SUMO-dependent phenotype.

The Arabidopsis SUMO E3 ligase SIZ1 mediates the temperature dependent trade-off between plant immunity and growth.

Increased ambient temperature is inhibitory to plant immunity including auto-immunity. SNC1-dependent auto-immunity is, for example, fully suppressed at 28°C. We found that the Arabidopsis sumoylation mutant siz1 displays SNC1-dependent auto-immunity at 22°C but also at 28°C, which was EDS1 dependent at both temperatures. This siz1 auto-immune phenotype provided enhanced resistance to Pseudomonas at both temperatures. Moreover, the rosette size of siz1 recovered only weakly at 28°C, while this temperature fully rescues the growth defects of other SNC1-dependent auto-immune mutants. This thermo-insensitivity of siz1 correlated with a compromised thermosensory growth response, which was independent of the immune regulators PAD4 or SNC1. Our data reveal that this high temperature induced growth response strongly depends on COP1, while SIZ1 controls the amplitude of this growth response. This latter notion is supported by transcriptomics data, i.e. SIZ1 controls the amplitude and timing of high temperature transcriptional changes including a subset of the PIF4/BZR1 gene targets. Combined our data signify that SIZ1 suppresses an SNC1-dependent resistance response at both normal and high temperatures. At the same time, SIZ1 amplifies the dark and high temperature growth response, likely via COP1 and upstream of gene regulation by PIF4 and BRZ1.

Arabidopsis TCP Transcription Factors Interact with the SUMO Conjugating Machinery in Nuclear Foci.

In Arabidopsis more than 400 proteins have been identified as SUMO targets, both in vivo and in vitro. Among others, transcription factors (TFs) are common targets for SUMO conjugation. Here we aimed to exhaustively screen for TFs that interact with the SUMO machinery using an arrayed yeast two-hybrid library containing more than 1,100 TFs. We identified 76 interactors that foremost interact with the SUMO conjugation enzyme SCE1 and/or the SUMO E3 ligase SIZ1. These interactors belong to various TF families, which control a wide range of processes in plant development and stress signaling. Amongst these interactors, the TCP family was overrepresented with several TCPs interacting with different proteins of the SUMO conjugation cycle. For a subset of these TCPs we confirmed that the catalytic site of SCE1 is essential for this interaction. In agreement, TCP1, TCP3, TCP8, TCP14, and TCP15 were readily SUMO modified in an E. coli sumoylation assay. Strikingly, these TCP-SCE1 interactions were found to redistribute these TCPs into nuclear foci/speckles, suggesting that these TCP foci represent sites for SUMO (conjugation) activity.

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.

Nuclear dynamics and genetic rearrangement in heterokaryotic colonies of Fusarium oxysporum.

Recent studies have shown horizontal transfer of chromosomes to be a potential key contributor to genome plasticity in asexual fungal pathogens. However, the mechanisms behind horizontal chromosome transfer in eukaryotes are not well understood. Here we investigated the role of conidial anastomosis in heterokaryon formation between incompatible strains of Fusarium oxysporum and determined the importance of heterokaryons for horizontal chromosome transfer. Using live-cell imaging we demonstrate that conidial pairing of incompatible strains under carbon starvation can result in the formation of viable heterokaryotic hyphae in F. oxysporum. Nuclei of the parental lines presumably fuse at some stage as conidia with a single nucleus harboring both marker histones (GFP- and RFP-tagged) are produced. Upon colony formation, this hybrid offspring is subject to progressive and gradual genome rearrangement. The parental genomes appear to become spatially separated and RFP-tagged histones, deriving from one of the strains, Fol4287, are eventually lost. With a PCR-based method we showed that markers for most of the chromosomes of this strain are lost, indicating a lack of Fol4287 chromosomes. This leaves offspring with the genomic background of the other strain (Fo47), but in some cases together with one or two chromosomes from Fol4287, including the chromosome that confers pathogenicity towards tomato.

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.

Dynamics of the establishment of multinucleate compartments in Fusarium oxysporum.

Nuclear dynamics can vary widely between fungal species and between stages of development of fungal colonies. Here we compared nuclear dynamics and mitotic patterns between germlings and mature hyphae in Fusarium oxysporum. Using fluorescently labeled nuclei and live-cell imaging, we show that F. oxysporum is subject to a developmental transition from a uninucleate to a multinucleate state after completion of colony initiation. We observed a special type of hypha that exhibits a higher growth rate, possibly acting as a nutrient scout. The higher growth rate is associated with a higher nuclear count and mitotic waves involving 2 to 6 nuclei in the apical compartment. Further, we found that dormant nuclei of intercalary compartments can reenter the mitotic cycle, resulting in multinucleate compartments with up to 18 nuclei in a single compartment.