Targeted Gene Replacement in Fungal Pathogens via Agrobacterium tumefaciens- Mediated Transformation

Author(s):  
Rasmus John Normand Frandsen ◽  
Mette Frandsen ◽  
Henriette Giese
2020 ◽  
Vol 6 (4) ◽  
pp. 314
Author(s):  
Johannes W. Debler ◽  
Bernadette M. Henares

Sustainable crop production is constantly challenged by the rapid evolution of fungal pathogens equipped with an array of host infection strategies and survival mechanisms. One of the devastating fungal pathogens that infect lentil is the ascomycete Ascochyta lentis which causes black spot or ascochyta blight (AB) on all above ground parts of the plant. In order to explore the mechanisms involved in the pathogenicity of A. lentis, we developed a targeted gene replacement method using Agrobacterium tumefaciens mediated transformation (ATMT) to study and characterize gene function. In this study, we investigated the role of scytalone dehydratase (SCD) in the synthesis of 1,8-dihydroxynaphthalene (DHN)-melanin in AlKewell. Two SCD genes have been identified in AlKewell, AlSCD1 and AlSCD2. Phylogenetic analysis revealed that AlSCD1 clustered with the previously characterized fungal SCDs; thus, AlSCD1 was disrupted using the targeted gene replacement vector, pTAR-hyg-SCD1. The vector was constructed in a single step process using Gibson Assembly, which facilitated an easy and seamless assembly of multiple inserts. The resulting AlKewell scd1::hyg transformants appeared light brown/brownish-pink in contrast to the dark brown pycnidia of the WT strain and ectopic transformant, indicating an altered DHN-melanin production. Disruption of AlSCD1 gene did not result in a change in the virulence profile of AlKewell towards susceptible and resistant lentil varieties. This is the first report of a targeted gene manipulation in A. lentis which serves as a foundation for the functional gene characterization to provide a better understanding of molecular mechanisms involved in pathogen diversity and host specificity.


2010 ◽  
Vol 23 (4) ◽  
pp. 366-375 ◽  
Author(s):  
Wende Liu ◽  
Shiyong Xie ◽  
Xinhua Zhao ◽  
Xin Chen ◽  
Wenhui Zheng ◽  
...  

Magnaporthe oryzae starts its infection by the attachment of pyriform conidia on rice tissues, and severity of the disease epidemic is proportional to the quantity of conidia produced in the rice blast lesions. However, the mechanism of conidial production is not well understood. Homeodomain proteins play critical roles in regulating various growth and developmental processes in fungi and other eukaryotes. Through targeted gene replacement, we find that deletion of HTF1, one of seven homeobox genes in the fungal genome, does not affect mycelial growth but causes total defect of conidial production. Further observation revealed that the Δhtf1 mutant produces significantly more conidiophores, which curve slightly near the tip but could not develop sterigmata-like structures. Although the Δhtf1 mutant fails to form conidia, it could still develop melanized appressoria from hyphal tips and infect plants. The expression level of HTF1 is significantly reduced in the Δmgb1 G-β and ΔcpkA deletion mutant, and the ACR1 but not CON7 gene that encodes transcription factor required for normal conidiogenesis is significantly downregulated in the Δhtf1 mutant. These data suggest that the HTF1 gene is essential for conidiogenesis, and may be functionally related to the trimeric G-protein signaling and other transcriptional regulators that are known to be important for conidiation in M. oryzae.


2006 ◽  
Vol 72 (11) ◽  
pp. 7111-7122 ◽  
Author(s):  
Olga V. Mavrodi ◽  
Dmitri V. Mavrodi ◽  
David M. Weller ◽  
Linda S. Thomashow

ABSTRACT Pseudomonas fluorescens Q8r1-96 produces 2,4-diacetylphloroglucinol (2,4-DAPG), a polyketide antibiotic that suppresses a wide variety of soilborne fungal pathogens, including Gaeumannomyces graminis var. tritici, which causes take-all disease of wheat. Strain Q8r1-96 is representative of the D-genotype of 2,4-DAPG producers, which are exceptional because of their ability to aggressively colonize and maintain large populations on the roots of host plants, including wheat, pea, and sugar beet. In this study, three genes, an sss recombinase gene, ptsP, and orfT, which are important in the interaction of Pseudomonas spp. with various hosts, were investigated to determine their contributions to the unusual colonization properties of strain Q8r1-96. The sss recombinase and ptsP genes influence global processes, including phenotypic plasticity and organic nitrogen utilization, respectively. The orfT gene contributes to the pathogenicity of Pseudomonas aeruginosa in plants and animals and is conserved among saprophytic rhizosphere pseudomonads, but its function is unknown. Clones containing these genes were identified in a Q8r1-96 genomic library, sequenced, and used to construct gene replacement mutants of Q8r1-96. Mutants were characterized to determine their 2,4-DAPG production, motility, fluorescence, colony morphology, exoprotease and hydrogen cyanide (HCN) production, carbon and nitrogen utilization, and ability to colonize the rhizosphere of wheat grown in natural soil. The ptsP mutant was impaired in wheat root colonization, whereas mutants with mutations in the sss recombinase gene and orfT were not. However, all three mutants were less competitive than wild-type P. fluorescens Q8r1-96 in the wheat rhizosphere when they were introduced into the soil by paired inoculation with the parental strain.


2003 ◽  
Vol 163 (3) ◽  
pp. 469-475 ◽  
Author(s):  
Satoru Yamazaki ◽  
Ryo Iwamoto ◽  
Kazuko Saeki ◽  
Masanori Asakura ◽  
Seiji Takashima ◽  
...  

Heparin-binding EGF-like growth factor (HB-EGF) is first synthesized as a membrane-anchored form (proHB-EGF), and its soluble form (sHB-EGF) is released by ectodomain shedding from proHB-EGF. To examine the significance of proHB-EGF processing in vivo, we generated mutant mice by targeted gene replacement, expressing either an uncleavable form (HBuc) or a transmembrane domain–truncated form (HBΔtm) of the molecule. HBuc/uc mice developed severe heart failure and enlarged heart valves, phenotypes similar to those in proHB-EGF null mice. On the other hand, mice carrying HBΔtm exhibited severe hyperplasia in both skin and heart. These results indicate that ectodomain shedding of proHB-EGF is essential for HB-EGF function in vivo, and that this process requires strict control.


2012 ◽  
Vol 7 (2) ◽  
pp. 247-255 ◽  
Author(s):  
Anne-Christin Graeser ◽  
Patricia Huebbe ◽  
Niels Storm ◽  
Wolfgang Höppner ◽  
Frank Döring ◽  
...  

2015 ◽  
Vol 112 (12) ◽  
pp. 2543-2549 ◽  
Author(s):  
Takayuki Arazoe ◽  
Kennosuke Miyoshi ◽  
Tohru Yamato ◽  
Tetsuo Ogawa ◽  
Shuichi Ohsato ◽  
...  

1991 ◽  
Vol 88 (16) ◽  
pp. 7170-7174 ◽  
Author(s):  
A. Cruz ◽  
C. M. Coburn ◽  
S. M. Beverley

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