1.An efficient genetic manipulation protocol for Ustilago esculenta.
Yu J1, Zhang Y1, Cui H1, Hu P1, Yu X1, Ye Z2. FEMS Microbiol Lett. 2015 Jun;362(12):fnv087. doi: 10.1093/femsle/fnv087. Epub 2015 Jun 1.
Ustilago esculenta grows within the flowering stem of the aquatic grass Zizania latifolia, resembling a fungal endophyte. The fungus colonizes Z. latifolia and induces swelling which results in the formation of galls near the base of the plant. Due to their unique flavor and textures these galls are considered as a delicacy in southern China. Efficient genetic manipulation is required to determine the relationship between U. esculenta and Z. latifolia. In this study, we report a protoplast-based transformation system for this unique fungal species. We have explored various factors (enzyme digesting conditions, osmotic pressure stabilizers, vectors and selection agents) that might impact protoplast yield and high frequencies of transformation. A haploid strain (UeT55) of U. esculenta was found to produce higher yields of protoplasts when treating with 15 mg mL(-1) lywallzyme in a sucrose-containing solution at 30°C for 3 h. The transformation frequencies were higher when fungal strain was transformed with a linear plasmid harboring hygromycin or carboxin resistance gene and regenerated on a sucrose-containing medium.
2.Red fluorescent proteins for imaging Zymoseptoria tritici during invasion of wheat.
Schuster M1, Kilaru S1, Guo M1, Sommerauer M2, Lin C3, Steinberg G4. Fungal Genet Biol. 2015 Jun;79:132-40. doi: 10.1016/j.fgb.2015.03.025.
The use of fluorescent proteins (FPs) in plant pathogenic fungi provides valuable insight into their intracellular dynamics, cell organization and invasion mechanisms. Compared with green-fluorescent proteins, their red-fluorescent "cousins" show generally lower fluorescent signal intensity and increased photo-bleaching. However, the combined usage of red and green fluorescent proteins allows powerful insight in co-localization studies. Efficient signal detection requires a bright red-fluorescent protein (RFP), combined with a suitable corresponding filter set. We provide a set of four vectors, suitable for yeast recombination-based cloning that carries mRFP, TagRFP, mCherry and tdTomato. These vectors confer carboxin resistance after targeted single-copy integration into the sdi1 locus of Zymoseptoria tritici. Expression of the RFPs does not affect virulence of this wheat pathogen. We tested all four RFPs in combination with four epi-fluorescence filter sets and in confocal laser scanning microscopy, both in and ex planta.
3.A gene locus for targeted ectopic gene integration in Zymoseptoria tritici.
Kilaru S1, Schuster M2, Latz M2, Das Gupta S2, Steinberg N3, Fones H2, Gurr SJ2, Talbot NJ2, Steinberg G4. Fungal Genet Biol. 2015 Jun;79:118-24. doi: 10.1016/j.fgb.2015.03.018.
Understanding the cellular organization and biology of fungal pathogens requires accurate methods for genomic integration of mutant alleles or fluorescent fusion-protein constructs. In Zymoseptoria tritici, this can be achieved by integrating of plasmid DNA randomly into the genome of this wheat pathogen. However, untargeted ectopic integration carries the risk of unwanted side effects, such as altered gene expression, due to targeting regulatory elements, or gene disruption following integration into protein-coding regions of the genome. Here, we establish the succinate dehydrogenase (sdi1) locus as a single "soft-landing" site for targeted ectopic integration of genetic constructs by using a carboxin-resistant sdi1(R) allele, carrying the point-mutation H267L. We use various green and red fluorescent fusion constructs and show that 97% of all transformants integrate correctly into the sdi1 locus as single copies. We also demonstrate that such integration does not affect the pathogenicity of Z.
4.Disruption of spindle checkpoint function in rats following 28 days of repeated administration of renal carcinogens.
Kimura M1, Mizukami S, Watanabe Y, Hasegawa-Baba Y, Onda N, Yoshida T, Shibutani M. J Toxicol Sci. 2016;41(1):91-104. doi: 10.2131/jts.41.91.
We previously reported that 28-day exposure to hepatocarcinogens that facilitate cell proliferation specifically alters the expression of G1/S checkpoint-related genes and proteins, induces aberrant early expression of ubiquitin D (UBD) at the G2 phase, and increases apoptosis in the rat liver, indicating G1/S and spindle checkpoint dysfunction. The present study aimed to determine the time of onset of carcinogen-specific cell-cycle disruption after repeated administration of renal carcinogens for up to 28 days. Rats were orally administered the renal carcinogens nitrofurantoin (NFT), 1-amino-2,4-dibromoantraquinone (ADAQ), and 1,2,3-trichloropropane (TCP) or the non-carcinogenic renal toxicants 1-chloro-2-propanol, triamterene, and carboxin for 3, 7 or 28 days. Both immunohistochemical single-molecule analysis and real-time RT-PCR analysis revealed that carcinogen-specific expression changes were not observed after 28 days of administration.