Obtaining of AtFUT knockout plants

  Figure 35: Fucosyltransferase 1 activity assay.  

High activity was observed with AtFUT1 which was transiently expressed in N. benthamiana leaves. The empty  vector p21 was included as a negative control. Substrate was tamarind xyloglucan. With AtFUT1 as the enzyme, a  conversion of unfucosylated XXLG into fucosylated XXFG of about 43.4 % could be detected. No XXFG could be  detected in the empty vector control p21.  

3.8 Study of knock-out or k nock-down plants affected in genes

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insertion point. Figure 36 gives an overview about the genes for which knockouts could be identified on gDNA level.

  Figure 36: Genomic DNA sequence of A. thaliana AtFUT candidates for which T‐DNA insertion lines could be  identified.  

Knockouts  could  be  identified  for  AtFUT1  (At2g03220),  AtFuT4  (At2g15390),  AtFUT5  (At1g15370),  AtFUT8  (At1g14100) and AtFUT10 (At2g15350). Information about genomic DNA structure has been obtained from TAIR. 

No information about 5’ and 3’ UTR was available for AtFUT5, AtFUT8 and AtFUT10. Except AtFUT10, all genes are  described having 2 exons and 1 intron. The T‐DNA insertion was predicted in promoter region for AtFUT4, AtFUT5  and AtFUT10 which could be confirmed. Insertion in an exon could be confirmed for AtFuT1 and AtFuT8. The  position of ATG and stop codon is indicated as well as the position of T‐DNA insertion flanking sequence. Magenta  arrows are primers used for screening of knockout plants. After sequencing, the insertion point has been evaluated  and its position is shown in base pairs downstream of the ATG or upstream in case of insertion in the promoter. 

For identification of knockout plants for AtFUT1 (At2g03220), 36 plants of SALK-line 101729 (N601729) were screened by PCR using the T-DNA insertion flanking primers AtFuT1-1F and AtFuT1-2R (Figure 37 A, lane 2) which gave a signal of 1.950 bp in the wildtype control. 2 plants were identified as putative knockouts of which one plant, designated Atfut1#15 has been selected and screened again using primers AtFuT-1F and LBa1 to confirm presence of T-DNA left border (Figure 37 B, lane 2). This screen has been repeated on 3 plants in the offspring generation using flanking primers (Figure 37 C, lanes 2 to 4). No signals were detected and one plant, designated Atfut1#15/1, was selected and screened again using primers for detection of the left border. A signal corresponding to the screen in the previous generation was observed (Figure 37 D, lane 2). This confirmed the homozygous knockout status on gDNA level and the plant was used for further investigations.

  Figure 37: Screening of knockout plants for AtFUT1.  

A) Screening for identification of homozygous knockouts using T‐DNA flanking primers, giving a signal of 1.950 bp  in the wildtype control (lane 3). No signal was observed in the knockout plant Atfut1#15 (lane 2). B) Confirmation  of presence of T‐DNA left border using one flanking‐ and a left border specific primer. A signal was achieved in  Atfut1#15. C) Repetition of screening in the offspring generation as described for A). No signals were achieved for  three screened plants (lanes 2 to 4), confirming the homozygous state, and an expected signal in the wildtype  control (lane 5, arrow). D) Offspring generation screened with a flanking‐ and a left border specific primer. A signal  was achieved in Atfut1#15/1. Lane 1 – Fermentas Mass Ruler DNA Ladder Mix and Fermentas 100 bp DNA Ladder  Plus. Col‐0 WT – Columbia wildtype was used as a control. 

For identification of knockout plants for AtFUT4 (At2g15390), 16 plants of the knockout line SALK_125310 (N625310) were screened using the T-DNA flanking primers AtFuT4-3F and AtFuT4-4R which gave a signal of 1 Kb in the wildtype control. 6 putative knockout plants, designated Atfut4, have been identified of which Atfut4#11 has been selected (Figure 38 A, lane 2). PCR screen using primers AtFuT4-3F and LBa1 confirmed presence of T-DNA left border (Figure 38 B, lane 2). This screen has been repeated in the offspring generation on 3 plants using flanking primers (Figure 38 C,

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lanes 2 to 4), and one plant, Atfut4#11/1, was selected and screened again using primers for detection of the left border. A signal corresponding to the screen in the previous generation was observed (Figure 38 D, lane 2). This confirmed the homozygous knockout status on gDNA level and the plant was used for further investigations.

  Figure 38: Screening of knockout plants for AtFUT4.  

A) Screening for identification of homozygous knockouts using T‐DNA flanking primers, giving a signal of 1.000 bp  in the wildtype control (lane 3). No signal was observed in the knockout plant Atfut4#11 (lane 2). B) Confirmation  of presence of T‐DNA left border using one flanking‐ and a left border specific primer. A signal was achieved in  Atfut4#11. C) Repetition of screening in the offspring generation as described for A). No signals were achieved for  three screened plants (lanes 2 to 4), confirming the homozygous state, and an expected signal in the wildtype  control (lane 5). D) Offspring generation screened with a flanking‐ and a left border specific primer. A signal was  achieved in Atfut4#11/1. Lane 1 – Fermentas Mass Ruler DNA Ladder Mix and Fermentas 100 bp DNA Ladder Plus. 

Col‐0 WT – Columbia wildtype was used as a control. 

For identification of knockout plants for AtFUT5 (At2g15370), two knockout lines, GK_872B07 (N317948 and N317969), were screened. No homozygous knockout candidates could be identified for line N317948. 13 plants of line N317969 were screened using the T-DNA flanking primers AtFuT5-3F and AtFuT5-4R which gave a signal of 1 Kb in the wildtype control, and 5 plants, designated Atfut5, were identified as putative knockout of which Atfut5#10 has been selected (Figure 39 A, lane 2). Presence of T-DNA left border was confirmed by using primers AtFuT5-3F and LB-GABI- KAT_new (Figure 39 B, lane 2). This screen has been repeated in the offspring generation on 3 plants using flanking primers (Figure 39 C, lanes 2 - 4), and one plant, Atfut5#10/1, was selected and screened again using primers for detection of left border.

A signal corresponding to the screen in the previous generation was observed (Figure 39 D, lane 2). This confirmed the homozygous knockout status on gDNA level and the plant was used for further investigations.

  Figure 39: Screening of knockout plants for AtFUT5.  

A) Screening for identification of homozygous knockouts using T‐DNA flanking primers, giving a signal of 1.000 bp  in the wildtype control (lane 3). No signal was observed in the knockout plant Atfut5#10 (lane 2). B) Confirmation  of presence of T‐DNA left border using one flanking‐ and a left border specific primer. A signal was achieved in  Atfut5#10. C) Repetition of screening in the offspring generation as described for A). No signals were achieved for  three screened plants (lanes 2 to 4), confirming the homozygous state, and an expected signal in the wildtype  control (lane 5). D) Offspring generation screened with a flanking‐ and a left border specific primer. A signal was  achieved in Atfut5#10/1. Lane 1 – Fermentas Mass Ruler DNA Ladder Mix and Fermentas 100 bp DNA Ladder Plus. 

Col‐0 WT – Columbia wildtype was used as a control. 

For identification of knockout plants of AtFUT8 (At1g14100), 17 plants of the knockout line SALK_010965 (N510965) were screened using T-DNA insertion flanking primers AtFuT8-1F and AtFuT8-2R which gave a signal of 1 KB in the wildtype control.

4 putative knockout plants, designated as Atfut8, were identified of which one plant, Atfut8#7, has been selected (Figure 40 A, lane 2). Screening with primers AtFuT8-1F and LBa1 confirmed presence of T-DNA left border (Figure 40 B, lane 2). This screen has been repeated in the offspring generation on 3 plants using flanking primers (Figure 40 C, lanes 2 - 4) and one plant, Atfut8#7/1, was selected and screened again using primers for detection of left border. A signal corresponding to the screen in the previous generation was observed (Figure 40 D, lane 2). This confirmed the homozygous knockout status on gDNA level and the plant was used for further investigations.

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  Figure 40: Screening of knockout plants for AtFUT8.  

A) Screening for identification of homozygous knockouts using T‐DNA flanking primers, giving a signal of 1.000 bp  in the wildtype control (lane 3). No signal was observed in the knockout plant Atfut8#7 (lane 2). B) Confirmation of  presence of T‐DNA left border using one flanking‐ and a left border specific primer. A signal was achieved in  Atfut8#7. C) Repetition of screening in the offspring generation as described for A). No signals were achieved for  three screened plants (lanes 2 to 4), confirming the homozygous state, and an expected signal in the wildtype  control (lane 5). D) Offspring generation screened with a flanking‐ and a left border specific primer. A signal was  achieved in Atfut8#7/1. Lane 1 – Fermentas Mass Ruler DNA Ladder Mix and Fermentas 100 bp DNA Ladder Plus. 

Col‐0 WT – Columbia wildtype was used as a control. 

Since AtFUT10 (At2g15350) has already been described as a confirmed homozygous line, 3 plants of line SALK_020408 (N859950) were screened using T- DNA flanking primers AtFuT10-3F and AtFuT10-4R which gave a signal of 1 Kb in the wildtype control. All 3 plants, designed as Atfut10, were identified as putative knockout plants and one plant, Atfut10#1, was selected (Figure 41 A, lane 2). Left border of T- DNA was confirmed by PCR screen using AtFuT10-3F and LBa1 and AtFuT10-4R and LBa1 (Figure 41 B, lane 2). This screen has been repeated in the offspring generation on 3 plants using flanking primers (Figure 41 C, lanes 2- 4). One plant, Atfut10#1/1, was selected and screened again with primers specific for left border. A signal corresponding to the screen in the previous generation was observed (Figure 41 D, lane 2). This confirmed the homozygous knockout status on gDNA level and the plant was used for further investigations.

  Figure 41: Screening of knockout plants for AtFUT10.  

A) Screening for identification of homozygous knockouts using T‐DNA flanking primers, giving a signal of 700 bp in  the wildtype control (lane 3). No signal was observed in the knockout plant Atfut10#1 (lane 2). B) Confirmation of  presence of T‐DNA left border using one flanking‐ and a left border specific primer. A signal was achieved in  Atfut10#1 (lane 2, arrow). C) Repetition of screening in the offspring generation as described for A). No signals  were achieved for three screened plants (lanes 2 to 4), confirming the homozygous state, and an expected signal in  the wildtype control (lane 5). D) Offspring generation screened with a flanking‐ and a left border specific primer. A  signal was achieved in Atfut10#1/1. Lane 1 – Fermentas 100 bp DNA Ladder Plus. Col‐0 WT – Columbia wildtype  was used as a control. 

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No knockouts could be identified for AtFUT2, AtFUT3, AtFUT6 and AtFUT7 (Figure 42).

  Figure 42: Genomic DNA sequence of A. thaliana AtFUT candidates for which no T‐DNA insertion lines could be  identified.  

No knockouts could be identified for AtFUT2 (At2g03210), AtFUT3 (At1g74420), AtFUT6 (At1g14080) and AtFUT7  (At1g14070). Information about genomic DNA structure has been obtained from TAIR. No information about 5’ 

and 3’ UTR was available for AtFUT2 and AtFUT7. All genes are described having 2 exons and 1 intron. The T‐DNA  insertion was predicted in promoter region for AtFUT3 and in an exon for AtFUT2, AtFUT6 and AtFUT7. The  position of ATG and stop codon is indicated as well as the position of T‐DNA insertion flanking sequence. Magenta  arrows are primers used for screening of knockout plants.  

For identification of knockout plants of AtFUT2 (At2g03210), 18 plants of the knockout line SALK_027979 (N527979) were screened using T-DNA insertion flanking primers AtFuT2-1F and AtFuT2-2R but no homozygous knockout candidates could be identified. Now it was suggested that these plants could be heterozygous instead of homozygous, therefore it was tried to screen for presence of T-DNA. No candidates could be identified with primers AtFuT2-1F and LBa1 and AtFuT2-2R and LBa1.

For identification of knockout plants of AtFUT3 (At1g74420), 35 plants of knockout line SALK_073920 (N573929) were screened using T-DNA insertion flanking primers AtFuT3-1F and AtFuT3-2R as well as AtFuT3-3F and AtFuT3-2R, and also here no homozygous knockout candidates could be found. No heterozygous plants could be found using primers AtFuT3-1F and LBa1 and AtFuT3-2R and LBa1.

For identification of knockout plants of AtFUT6 (At1g14080), 29 plants of knockout line SALK_078348 (N578348) were screened using T-DNA insertion flanking primers AtFuT6-1F and AtFuT6-2R as well as AtFuT6-3F and AtFuT6-4R but no homozygous knockout candidates could be found. No heterozygous plants could be found using primers AtFuT6-3F and LBa1 and AtFuT6-4R and LBa1.

For identification of knockout plants of AtFUT7 (At1g14070), 42 plants of knockout line SALK_049570 (N859993) were screened using T-DNA insertion flanking primers AtFuT7-1F and AtFuT7-2R as well as AtFuT7-3F and AtFuT7-4R but no homozygous knockout candidates could be found. No heterozygous plants could be found using primers AtFuT7-3F and LBa1 and AtFuT7-4R and LBa1.