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UvHOG1 is important for hyphal growth and stress responses in the rice false smut fungus Ustilaginoidea virens.

Zheng D, Wang Y, Han Y, Xu JR, Wang C - Sci Rep (2016)

Bottom Line: Deletion of UvHOG1 resulted in reduced expression of the stress response-related genes UvATF1 and UvSKN7.In the Uvhog1 mutant, NaCl treatment failed to stimulate the accumulation of sorbitol and glycerol.In addition, the Uvhog1 mutant had reduced toxicity on shoot growth in rice seed germination assays.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China.

ABSTRACT
Rice false smut caused by Ustilaginoidea virens is one of the most important diseases of rice worldwide. Although its genome has been sequenced, to date there is no report on targeted gene deletion in U. virens and no molecular studies on genetic mechanisms regulating the infection processes of this destructive pathogen. In this study, we attempted to generate knockout mutants of the ortholog of yeast HOG1 MAP kinase gene in U. virens. One Uvhog1 deletion mutant was identified after screening over 600 hygromycin-resistant transformants generated by Agrobacterium tumefaciens mediated transformation. The Uvhog1 mutant was reduced in growth rate and conidiation but had increased sensitivities to SDS, Congo red, and hyperosmotic stress. Deletion of UvHOG1 resulted in reduced expression of the stress response-related genes UvATF1 and UvSKN7. In the Uvhog1 mutant, NaCl treatment failed to stimulate the accumulation of sorbitol and glycerol. In addition, the Uvhog1 mutant had reduced toxicity on shoot growth in rice seed germination assays. Overall, as the first report of targeted gene deletion mutant in U. virens, our results showed that UvHOG1 likely has conserved roles in regulating stress responses, hyphal growth, and possibly secondary metabolism.

No MeSH data available.


Related in: MedlinePlus

Generation of the Uvhog1 mutant.(A) The UvHOG1 locus and gene replacement construct. The UvHOG1 and hph genes are marked with empty and black arrows, respectively. 1F, NF, 2R, 3F, 4R, 5F, 6R, and NR are primers used to amplify the flanking sequences or for mutant screening. B: BamHI. LB: Left border; RB: Right border. (B) Southern blots of genomic DNA isolated from the wild type strain UV-8b, Uvhog1 mutant M1, and an ectopic transformant M2 were hybridized with probe A (left) amplified with primers 5F and 6R or probe B (right) amplified with primers H852 and H850. All DNA samples were digested with BamHI. (C) Colony morphology of the wild type, Uvhog1 mutant, and complementary strain grown on 5xYEG plates.
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f1: Generation of the Uvhog1 mutant.(A) The UvHOG1 locus and gene replacement construct. The UvHOG1 and hph genes are marked with empty and black arrows, respectively. 1F, NF, 2R, 3F, 4R, 5F, 6R, and NR are primers used to amplify the flanking sequences or for mutant screening. B: BamHI. LB: Left border; RB: Right border. (B) Southern blots of genomic DNA isolated from the wild type strain UV-8b, Uvhog1 mutant M1, and an ectopic transformant M2 were hybridized with probe A (left) amplified with primers 5F and 6R or probe B (right) amplified with primers H852 and H850. All DNA samples were digested with BamHI. (C) Colony morphology of the wild type, Uvhog1 mutant, and complementary strain grown on 5xYEG plates.

Mentions: Because Agrobacterium tumafaciens-mediated transformation (ATMT) is known to increase the frequency of homologous recombination27, we then cloned the UvHOG1 gene replacement construct generated by double-joint PCR into the binary vector pCAMBIA130028. The resulting vector was introduced into A. tumafaciens and used to transform conidia of UV-8b. A total of 619 hygromycin-resistant ATMT transformants were screened by PCR with primers (see Supplementary Table S1 online) located in the deleted region. One putative knockout mutant M1 was identified (Fig. 1A) and further confirmed by Southern blot hybridization (Fig. 1B). In the wild type and ectopic transformant M2, a 3.5-kb BamHI band was detected with an UvHOG1 fragment as the probe (Fig. 1B). The same probe had no hybridization signals in transformant M1. When hybridized with a fragment of the hph gene, a 3.2-kb band of the expected size derived from the gene replacement event was detected in the Uvhog1 deletion mutant M1 (Fig. 1B). The wild type had no hybridization signals but ectopic transformant M2 had a 5.0-kb band (Fig. 1B). Therefore, the homologous recombination efficiency was as low as 0.16% for the UvHOG1 gene in U. virens.


UvHOG1 is important for hyphal growth and stress responses in the rice false smut fungus Ustilaginoidea virens.

Zheng D, Wang Y, Han Y, Xu JR, Wang C - Sci Rep (2016)

Generation of the Uvhog1 mutant.(A) The UvHOG1 locus and gene replacement construct. The UvHOG1 and hph genes are marked with empty and black arrows, respectively. 1F, NF, 2R, 3F, 4R, 5F, 6R, and NR are primers used to amplify the flanking sequences or for mutant screening. B: BamHI. LB: Left border; RB: Right border. (B) Southern blots of genomic DNA isolated from the wild type strain UV-8b, Uvhog1 mutant M1, and an ectopic transformant M2 were hybridized with probe A (left) amplified with primers 5F and 6R or probe B (right) amplified with primers H852 and H850. All DNA samples were digested with BamHI. (C) Colony morphology of the wild type, Uvhog1 mutant, and complementary strain grown on 5xYEG plates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837404&req=5

f1: Generation of the Uvhog1 mutant.(A) The UvHOG1 locus and gene replacement construct. The UvHOG1 and hph genes are marked with empty and black arrows, respectively. 1F, NF, 2R, 3F, 4R, 5F, 6R, and NR are primers used to amplify the flanking sequences or for mutant screening. B: BamHI. LB: Left border; RB: Right border. (B) Southern blots of genomic DNA isolated from the wild type strain UV-8b, Uvhog1 mutant M1, and an ectopic transformant M2 were hybridized with probe A (left) amplified with primers 5F and 6R or probe B (right) amplified with primers H852 and H850. All DNA samples were digested with BamHI. (C) Colony morphology of the wild type, Uvhog1 mutant, and complementary strain grown on 5xYEG plates.
Mentions: Because Agrobacterium tumafaciens-mediated transformation (ATMT) is known to increase the frequency of homologous recombination27, we then cloned the UvHOG1 gene replacement construct generated by double-joint PCR into the binary vector pCAMBIA130028. The resulting vector was introduced into A. tumafaciens and used to transform conidia of UV-8b. A total of 619 hygromycin-resistant ATMT transformants were screened by PCR with primers (see Supplementary Table S1 online) located in the deleted region. One putative knockout mutant M1 was identified (Fig. 1A) and further confirmed by Southern blot hybridization (Fig. 1B). In the wild type and ectopic transformant M2, a 3.5-kb BamHI band was detected with an UvHOG1 fragment as the probe (Fig. 1B). The same probe had no hybridization signals in transformant M1. When hybridized with a fragment of the hph gene, a 3.2-kb band of the expected size derived from the gene replacement event was detected in the Uvhog1 deletion mutant M1 (Fig. 1B). The wild type had no hybridization signals but ectopic transformant M2 had a 5.0-kb band (Fig. 1B). Therefore, the homologous recombination efficiency was as low as 0.16% for the UvHOG1 gene in U. virens.

Bottom Line: Deletion of UvHOG1 resulted in reduced expression of the stress response-related genes UvATF1 and UvSKN7.In the Uvhog1 mutant, NaCl treatment failed to stimulate the accumulation of sorbitol and glycerol.In addition, the Uvhog1 mutant had reduced toxicity on shoot growth in rice seed germination assays.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China.

ABSTRACT
Rice false smut caused by Ustilaginoidea virens is one of the most important diseases of rice worldwide. Although its genome has been sequenced, to date there is no report on targeted gene deletion in U. virens and no molecular studies on genetic mechanisms regulating the infection processes of this destructive pathogen. In this study, we attempted to generate knockout mutants of the ortholog of yeast HOG1 MAP kinase gene in U. virens. One Uvhog1 deletion mutant was identified after screening over 600 hygromycin-resistant transformants generated by Agrobacterium tumefaciens mediated transformation. The Uvhog1 mutant was reduced in growth rate and conidiation but had increased sensitivities to SDS, Congo red, and hyperosmotic stress. Deletion of UvHOG1 resulted in reduced expression of the stress response-related genes UvATF1 and UvSKN7. In the Uvhog1 mutant, NaCl treatment failed to stimulate the accumulation of sorbitol and glycerol. In addition, the Uvhog1 mutant had reduced toxicity on shoot growth in rice seed germination assays. Overall, as the first report of targeted gene deletion mutant in U. virens, our results showed that UvHOG1 likely has conserved roles in regulating stress responses, hyphal growth, and possibly secondary metabolism.

No MeSH data available.


Related in: MedlinePlus