Paper · 2014 · Zhongguo nongye Kexue

Molecular Cloning of T-DNA Targeted Genes of Ustilaginoidea virens Pathogenicity-Defective Mutant Strain B-1015

Huang Le

13
claims
26
verbatim quotes
0
citations
2
research areas
—
Not open access

Abstract

【Objective】 The objective of this study is to analyze the biological phenotypes of the pathogenicity-defective mutant strain B-1015, to clone its T-DNA integration flanking sequences, and finally to reveal the functions of targeted genes in pathogenesis of Ustilaginoidea virens. The study will contribute to better understanding of the molecular pathogenesis of U. virens. 【Method】 Biological phenotypes of B-1015 were analyzed by testing growth rate, sporulation ability, spore germination rate and pathogenicity. The copy numbers of T-DNA inserted into B-1015 were identified by Southern blot. The flanking sequences of T-DNA were cloned by hiTail-PCR and the whole gene sequences of the T-DNA targeted genes were cloned by RACE PCR. The genes expression were detected by RT-PCR. 【Result】By comparing with wild type strain P1, the sporulation ability and growth rate of the B-1015 were significantly declined, but the spore germination rate had no significant difference. The inserted mutant lost its pathogenicity in rice. Molecular detection analysis confirmed that the B-1015 was a stable single T-DNA insertional mutant. The T-DNA flanking sequences were obtained by hiTail-PCR. Two genes, Uvt-1015R and Uvt-1015L, were cloned by RACE PCR. Gene sequence analysis showed that Uvt-1015R contained a 948 bp open reading frame, which could encode a 295 amino acid protein, a 341 bp 5′-UTR and a 271 bp 3′-UTR. Another gene, Uvt-1015L, comprised a 351 bp open reading frame coding a 116 amino acid protein, a 31 bp 5′-UTR and a 174 bp 3′-UTR. By searching in the known protein, the Uvt-1015R and Uvt-1015L genes showed no homology of any protein. Sequence alignment showed that the flanking sequences of T-DNA were non-adjacent in the wild type genome and the T-DNA disrupted the two genes sequence. RT-PCR analysis confirmed that compared to P1 strain, the transcripts of Uvt-1015R was significantly decreased, and the transcripts of Uvt-1015L was not detected any more. 【Conclusion】 In B-1015, both the T-DNA insertion and chromosomal rearrangement caused important biological phenotype mutant, thus leading to the loss of pathogenicity.

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Claims 13

Observationobservedoriginal resultnegativesignificant (as stated)measured

Claim · model-written summary

B-1015 sporulation and growth rate declined significantly versus wild type P1.

Interactor
B-1015e1pathogen
Outcome
sporulation ability and growth rate of the B-1015e1
Direction
declinede1
Comparator
wild type strain P1e1

Organisms pictured · Representative pictures of the matched species, not figures from the paper.

Oryza sativa
Oryza sativa · Taxon · picture from its Wikimedia Commons categoryDesmanthus4food · CC BY-SA 3.0 us · Wikimedia Commons ↗

Verbatim

e0

“pathogenicity in rice”

✓ located verbatim in the source · abstract, characters 1004–1025 · show in text ↑
e1

“By comparing with wild type strain P1, the sporulation ability and growth rate of the B-1015 were significantly declined”

✓ located verbatim in the source · abstract, characters 791–911 · show in text ↑
Observationobservedoriginal resultno effectnot significant (as stated)measured

Claim · model-written summary

B-1015 spore germination rate did not differ significantly from P1.

Interactor
B-1015e2pathogen
Outcome
spore germination ratee1
Direction
no significant differencee1

Organisms pictured · Representative pictures of the matched species, not figures from the paper.

Oryza sativa
Oryza sativa · Taxon · picture from its Wikimedia Commons categoryDesmanthus4food · CC BY-SA 3.0 us · Wikimedia Commons ↗

Verbatim

e0

“pathogenicity in rice”

✓ located verbatim in the source · abstract, characters 1004–1025 · show in text ↑
e1

“spore germination rate had no significant difference”

✓ located verbatim in the source · abstract, characters 921–973 · show in text ↑
e2

“growth rate of the B-1015 were significantly declined”

✓ located verbatim in the source · abstract, characters 858–911 · show in text ↑
Observationobservedoriginal resultpositivemeasured

Claim · model-written summary

The insertional mutant lost pathogenicity in rice.

Interactor
inserted mutante0pathogen
Outcome
pathogenicity in ricee0
Direction
loste0

Organisms pictured · Representative pictures of the matched species, not figures from the paper.

Oryza sativa
Oryza sativa · Taxon · picture from its Wikimedia Commons categoryDesmanthus4food · CC BY-SA 3.0 us · Wikimedia Commons ↗

Verbatim

e0

“The inserted mutant lost its pathogenicity in rice.”

✓ located verbatim in the source · abstract, characters 975–1026 · show in text ↑
Observationobservedoriginal resultmeasured

Claim · model-written summary

B-1015 was confirmed as a stable single T-DNA insertional mutant.

Interactor
B-1015e1pathogen
Outcome
stable single T-DNA insertional mutante1

Organisms pictured · Representative pictures of the matched species, not figures from the paper.

Oryza sativa
Oryza sativa · Taxon · picture from its Wikimedia Commons categoryDesmanthus4food · CC BY-SA 3.0 us · Wikimedia Commons ↗

Verbatim

e0

“pathogenicity in rice”

✓ located verbatim in the source · abstract, characters 1004–1025 · show in text ↑
e1

“the B-1015 was a stable single T-DNA insertional mutant”

✓ located verbatim in the source · abstract, characters 1071–1126 · show in text ↑
Observationobservedoriginal resultmeasured

Claim · model-written summary

T-DNA flanking sequences were obtained by hiTail-PCR.

Outcome
T-DNA flanking sequencese1

Organisms pictured · Representative pictures of the matched species, not figures from the paper.

Oryza sativa
Oryza sativa · Taxon · picture from its Wikimedia Commons categoryDesmanthus4food · CC BY-SA 3.0 us · Wikimedia Commons ↗

Verbatim

e0

“pathogenicity in rice”

✓ located verbatim in the source · abstract, characters 1004–1025 · show in text ↑
e1

“The T-DNA flanking sequences were obtained by hiTail-PCR.”

✓ located verbatim in the source · abstract, characters 1128–1185 · show in text ↑
Observationobservedoriginal resultmeasured

Claim · model-written summary

Uvt-1015R and Uvt-1015L were cloned by RACE PCR.

Gene
Uvt-1015R and Uvt-1015Le1
Outcome
cloned by RACE PCRe1

Organisms pictured · Representative pictures of the matched species, not figures from the paper.

Oryza sativa
Oryza sativa · Taxon · picture from its Wikimedia Commons categoryDesmanthus4food · CC BY-SA 3.0 us · Wikimedia Commons ↗

Verbatim

e0

“pathogenicity in rice”

✓ located verbatim in the source · abstract, characters 1004–1025 · show in text ↑
e1

“Two genes, Uvt-1015R and Uvt-1015L, were cloned by RACE PCR.”

✓ located verbatim in the source · abstract, characters 1186–1246 · show in text ↑
Observationobservedoriginal resultmeasured

Claim · model-written summary

Uvt-1015R had a 948 bp open reading frame encoding a 295 amino acid protein and 341 bp and 271 bp untranslated regions.

Gene
Uvt-1015Re1
Outcome
open reading framee1
Value
948 bpe1

Organisms pictured · Representative pictures of the matched species, not figures from the paper.

Oryza sativa
Oryza sativa · Taxon · picture from its Wikimedia Commons categoryDesmanthus4food · CC BY-SA 3.0 us · Wikimedia Commons ↗

Verbatim

e0

“pathogenicity in rice”

✓ located verbatim in the source · abstract, characters 1004–1025 · show in text ↑
e1

“Uvt-1015R contained a 948 bp open reading frame, which could encode a 295 amino acid protein, a 341 bp 5′-UTR and a 271 bp 3′-UTR.”

✓ located verbatim in the source · abstract, characters 1282–1412 · show in text ↑
Observationobservedoriginal resultmeasured

Claim · model-written summary

Uvt-1015L had a 351 bp open reading frame coding a 116 amino acid protein and 31 bp and 174 bp untranslated regions.

Gene
Uvt-1015Le1
Outcome
open reading framee1
Value
351 bpe1

Organisms pictured · Representative pictures of the matched species, not figures from the paper.

Oryza sativa
Oryza sativa · Taxon · picture from its Wikimedia Commons categoryDesmanthus4food · CC BY-SA 3.0 us · Wikimedia Commons ↗

Verbatim

e0

“pathogenicity in rice”

✓ located verbatim in the source · abstract, characters 1004–1025 · show in text ↑
e1

“Uvt-1015L, comprised a 351 bp open reading frame coding a 116 amino acid protein, a 31 bp 5′-UTR and a 174 bp 3′-UTR.”

✓ located verbatim in the source · abstract, characters 1427–1544 · show in text ↑
Observationobservedoriginal resultmeasured

Claim · model-written summary

Neither Uvt-1015R nor Uvt-1015L had homology to a known protein.

Gene
Uvt-1015R and Uvt-1015Le1
Outcome
no homology of any proteine1

Organisms pictured · Representative pictures of the matched species, not figures from the paper.

Oryza sativa
Oryza sativa · Taxon · picture from its Wikimedia Commons categoryDesmanthus4food · CC BY-SA 3.0 us · Wikimedia Commons ↗

Verbatim

e0

“pathogenicity in rice”

✓ located verbatim in the source · abstract, characters 1004–1025 · show in text ↑
e1

“Uvt-1015R and Uvt-1015L genes showed no homology of any protein”

✓ located verbatim in the source · abstract, characters 1584–1647 · show in text ↑
Observationobservedoriginal resultmeasured

Claim · model-written summary

T-DNA flanking sequences were nonadjacent in the wild type genome, and T-DNA disrupted two gene sequences.

Outcome
flanking sequences of T-DNAe1

Organisms pictured · Representative pictures of the matched species, not figures from the paper.

Oryza sativa
Oryza sativa · Taxon · picture from its Wikimedia Commons categoryDesmanthus4food · CC BY-SA 3.0 us · Wikimedia Commons ↗

Verbatim

e0

“pathogenicity in rice”

✓ located verbatim in the source · abstract, characters 1004–1025 · show in text ↑
e1

“the flanking sequences of T-DNA were non-adjacent in the wild type genome and the T-DNA disrupted the two genes sequence”

✓ located verbatim in the source · abstract, characters 1680–1800 · show in text ↑
Observationobservedoriginal resultnegativesignificant (as stated)measured

Claim · model-written summary

Uvt-1015R transcripts decreased significantly compared with P1.

Gene
Uvt-1015Re1
Outcome
transcripts of Uvt-1015Re1
Direction
decreasede1
Comparator
P1 straine1

Organisms pictured · Representative pictures of the matched species, not figures from the paper.

Oryza sativa
Oryza sativa · Taxon · picture from its Wikimedia Commons categoryDesmanthus4food · CC BY-SA 3.0 us · Wikimedia Commons ↗

Verbatim

e0

“pathogenicity in rice”

✓ located verbatim in the source · abstract, characters 1004–1025 · show in text ↑
e1

“compared to P1 strain, the transcripts of Uvt-1015R was significantly decreased”

✓ located verbatim in the source · abstract, characters 1833–1912 · show in text ↑
Observationobservedoriginal resultnegativemeasured

Claim · model-written summary

Uvt-1015L transcripts were no longer detected.

Gene
Uvt-1015Le1
Outcome
transcripts of Uvt-1015Le1
Direction
not detected any moree1

Organisms pictured · Representative pictures of the matched species, not figures from the paper.

Oryza sativa
Oryza sativa · Taxon · picture from its Wikimedia Commons categoryDesmanthus4food · CC BY-SA 3.0 us · Wikimedia Commons ↗

Verbatim

e0

“pathogenicity in rice”

✓ located verbatim in the source · abstract, characters 1004–1025 · show in text ↑
e1

“the transcripts of Uvt-1015L was not detected any more”

✓ located verbatim in the source · abstract, characters 1918–1972 · show in text ↑
Mechanismauthor interpretationoriginal resultpositivemeasured

Claim · model-written summary

The authors attributed B-1015 loss of pathogenicity to T-DNA insertion and chromosomal rearrangement.

Outcome
loss of pathogenicitye1
Qualifier
T-DNA insertion and chromosomal rearrangemente1

Organisms pictured · Representative pictures of the matched species, not figures from the paper.

Oryza sativa
Oryza sativa · Taxon · picture from its Wikimedia Commons categoryDesmanthus4food · CC BY-SA 3.0 us · Wikimedia Commons ↗

Verbatim

e0

“pathogenicity in rice”

✓ located verbatim in the source · abstract, characters 1004–1025 · show in text ↑
e1

“both the T-DNA insertion and chromosomal rearrangement caused important biological phenotype mutant, thus leading to the loss of pathogenicity”

✓ located verbatim in the source · abstract, characters 1998–2140 · show in text ↑