The Fusarium oxysporum effector Six6 contributes to virulence and suppresses I-2-mediated cell death.

Plant pathogens secrete effectors to manipulate their host and facilitate colonization. Fusarium oxysporum f. sp. lycopersici is the causal agent of Fusarium wilt disease in tomato. Upon infection, F. oxysporum f. sp. lycopersici secretes numerous small proteins into the xylem sap (Six proteins). Most Six proteins are unique to F. oxysporum, but Six6 is an exception; a homolog is also present in two Colletotrichum spp. SIX6 expression was found to require living host cells and a knockout of SIX6 in F. oxysporum f. sp. lycopersici compromised virulence, classifying it as a genuine effector. Heterologous expression of SIX6 did not affect growth of Agrobacterium tumefaciens in Nicotiana benthamiana leaves or susceptibility of Arabidopsis thaliana toward Verticillium dahliae, Pseudomonas syringae, or F. oxysporum, suggesting a specific function for F. oxysporum f. sp. lycopersici Six6 in the F. oxysporum f. sp. lycopersici- tomato pathosystem. Remarkably, Six6 was found to specifically suppress I-2-mediated cell death (I2CD) upon transient expression in N. benthamiana, whereas it did not compromise the activity of other cell-death-inducing genes. Still, this I2CD suppressing activity of Six6 does not allow the fungus to overcome I-2 resistance in tomato, suggesting that I-2-mediated resistance is independent from cell death.

The interplay between the GATA transcription factors AreA, the global nitrogen regulator and AreB in Fusarium fujikuroi.

Nitrogen metabolite repression (NMR) in filamentous fungi is controlled by the GATA transcription factors AreA and AreB. While AreA mainly acts as a positive regulator of NMR-sensitive genes, the role of AreB is not well understood. We report the characterization of AreB and its interplay with AreA in the gibberellin-producing fungus Fusarium fujikuroi. The areB locus produces three different transcripts that each code for functional proteins fully complementing the areB deletion mutant that influence growth and secondary metabolism. However, under nitrogen repression, the AreB isoforms differ in subcellular localization indicating distinct functions under these conditions. In addition, AreA and two isoforms of AreB colocalize in the nucleus under low nitrogen, but their nuclear localization disappears under conditions of high nitrogen. Using a bimolecular fluorescence complementation (BiFC) approach we showed for the first time that one of the AreB isoforms interacts with AreA when starved of nitrogen. Cross-species complementation revealed that some AreB functions are retained between F. fujikuroi and Aspergillus nidulans while others have diverged. By comparison to other fungi where AreB was postulated to function as a negative counterpart of AreA, AreB can act as both repressor and activator of transcription in F. fujikuroi.

Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori.

In this study, the efficiency of gene replacement in Aspergillus awamori between Agrobacterium-mediated transformation and CaCl(2)/PEG-mediated transformation was compared. For the genes, pyrG and gfaA, it was found that the homologous recombination frequencies obtained by Agrobacterium-mediated transformation were 3- to 6-fold higher than the frequencies obtained with CaCl(2)/PEG protoplast transformation. For the pyrG gene, it was found that Agrobacterium-mediated transformation allowed an efficient homologous recombination with shorter DNA flanks than CaCl(2)/PEG protoplast transformation. Finally, the addition of the dominant amdS marker as a second selection marker to the gene replacement cassette led to a further 2-fold enrichment in transformants with gene replacement events, resulting in a gene replacement frequency of 55%. Based on the data it can be concluded that Agrobacterium-mediated transformation is an efficient tool for gene replacement and that the amdS gene can be successfully used as a second selection marker to select transformants with putative gene replacement.

Development of a system for integrative and stable transformation of the zygomycete Rhizopus oryzae by Agrobacterium-mediated DNA transfer.

Two transformation systems, based on the use of CaCl(2)/PEG and Agrobacterium tumefaciens, respectively, were developed for the zygomycete Rhizopus oryzae. Irrespective of the selection marker used, a pyr4 marker derived from R. niveus or a dominant amdS(+) marker from Aspergillus nidulans, and irrespective of the configuration of the transforming DNA (linear or circular), the transformants obtained with the CaCl(2)/PEG transformation method were found to carry multiple copies of tandemly linked vector molecules, which failed to integrate into the genomic DNA. Furthermore, these transformants displayed low mitotic stability. In contrast, transformants obtained by Agrobacterium-mediated transformation were mitotically stable, even under non-selective conditions. Detailed analysis of these transformants revealed that the transforming DNA had integrated into the genome of R. oryzae at a single locus in independently obtained transformants. In addition, truncation of the transforming DNA was observed, resulting in the integration of the R. niveus pyr4 marker gene, but not the second gene located on the transferred DNA. Modification of the transforming DNA, resulting in partial resistance to restriction enzyme digestion, was observed in transformants obtained with the CaCl(2)/PEG transformation method, suggesting that a specific genome defence mechanism may exist in R. oryzae. It is likely that the unique mechanism used by A. tumefaciens to deliver its transferred DNA to its hosts facilitates bypass of the host defence mechanisms, thus allowing the DNA to integrate into the chromosomal genome.

The Aspergillus nidulans amdS gene as a marker for the identification of multicopy T-DNA integration events in Agrobacterium-mediated transformation of Aspergillus awamori.

The Aspergillus nidulans amdS selection marker was used for the identification of multicopy T-DNA insertions in Agrobacterium-mediated transformation of Asp. awamori. The selection of transformants on agar plates containing acetamide as sole nitrogen source and hygromycin resulted in a six-fold decrease in the transformation frequency, compared with the transformation frequency obtained after hygromycin selection alone. However, it was found that 47% of the transformants obtained after hygromycin and acetamide double selection contained multiple T-DNA integrations. Furthermore, it was found that the multicopy transformants could easily be identified based on their growth rate on agar plates containing acetamide medium. Based on these data, it can be concluded that the amdS marker can also be used as a selection marker in Agrobacterium-mediated transformation of Asp. awamori and that it is a very useful marker to identify those transformants containing multiple T-DNA integrations.

Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori.

The Agrobacterium-mediated transformation of Aspergillus awamori was optimized using defined co-cultivation conditions, which resulted in a reproducible and efficient transformation system. Optimal co-cultivation conditions were used to study the role of Agrobacterium tumefaciens virulence proteins in T-DNA transfer. This study revealed that inactivation of either of the regulatory proteins (VirA, VirG), any of the transport pore proteins (VirB), proteins involved in generation of the T-strand (VirD, VirC) or T-strand protection and targeting (VirE2) abolishes or severely reduces the formation of transformants. The results indicate that the Agrobacterium-mediated transformation of A. awamori requires an intact T-DNA machinery for efficient transformation; however, the plant host range factors, like VirE3, VirH, and VirF, are not important.