J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 1 Appendix 5

Efficacy and Tissue

- Specificity of Gene Silencing

INDEX

Section Page

Index 1

Introduction 2

Results 3

Summary 31

Materials and Methods 33

Conclusions 35

References 37

Certification 3 8J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 2 INTRODUCTION

Expression of the four genes targeted for gene silencing by transformation with pSIM1278 was tested in
tubers

, stolons, leaves, stems, roots, and flowers of the potatoes. The purpose of these studies was to
show the effectiveness of gene silencing based on RNA expression as measured through Northern blot analyses. It’s important to note that interpretation of the Northern blots requires judgment and the reported results represent a qualitative approach whereby the genes were considered downregulated.
All down

- regulated genes indicated in the summary ( Table 1) appeared reduced when compared with the respective controls , without differentiation between re duction levels, as appropriate for a qualitative method . When both greenhouse and field tubers were analyzed, a judgment was made after reviewing both gels.

Considering the potato events in this study , expression of the targeted genes would provide eviden ce that the desired phenotype with low asparagine, reduced black spot bruise , and lowered reducing sugars had been achieved. It could provide information relevant to the plant pest risk assessment, for example, all Innate TM potatoes in the study had reduced or similar expression to the controls. Thus, the altered expression is unlikely to affect weediness or other plant pest characteristics. Also, with these events, the expression studies pertain to endogenous proteins rat her than new proteins that could require a safety evaluation, such as those associated with herbicide tolerance or insect resistance. Gene silencing methods were employed to lower the activity of the Asn1, Ppo5, PhL, and R1 native proteins, and transcrip t levels rather than protein amounts were evaluated to link new phenotypic traits to changes at the molecular level. For this purpose, RNA was isolated from various tissues, and the RNA was used to carry out Northern blot analyses.

The promoters used to drive expression of the two inverted repeats, the Agp promoter and the Gbss promoter, have been well characterized and are known to be highly active in tubers and stolons while
induc ing some transcription in photosynthetically

- active tissues and roots (Nak ata et al . 1994; Visser et al

. 1991)

. Therefore, silencing effects mediated by expression of the inverted repeats were predicted to be strong in tubers and stolons, and less pronounced in leaves, stems, roots, and flowers. The silencing
effects in tissues other than tubers and stolons may have been further accentuated by the anticipated mobility of small RNAs produced by the silencing cassettes (Molnar et al

. 2010).

Northern blot analyses were carried out t o determine the actual effects of the transforma tions on expression levels of the four targeted genes in a tissue

- specific manner for each event

.
Most studies were carried out on tissues from greenhouse

- grown material including tubers, stolons, roots, stems, leaves, and flowers

. Additionally, some fiel d

- grown tubers were analyzed as well because tubers are the target tissue s for down

- regulation of the four genes.J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 3 RESULTS

Transcript Levels in Tubers

The transcript levels of target genes in various tissues of untransformed plants and the events were determined by Northern blot analysis.

In tubers of untransformed controls as viewed by Northern blot analyses , transcript levels were higher compared with the events for the Asn1
, Ppo5
, PhL

, and R1 genes. A comparison of northern blots
indicated that the A sn1
, Ppo5 , and PhL were expressed at similar levels in tubers from greenhouse ( Figures 1A, 2A, 3A and 4A ) and field ( Figures 1B, 2B, 3B and 4B )

. One exception was observed with Ppo5 silencing, in which greenhouse

- grown tubers had higher expression levels in both controls and events than in tubers from the field ( Figure 2B ). In contrast, the R1 gene was only partially silenced , with slight differences observed between events and contr ols in either greenhouse

- grown or tubers from the
field.

Strongly reduced transcript levels for the Asn1 and Ppo5 genes in tubers of all events were intentional
and are associated with low

- acrylamide potential and black spot bruise tolerance ( Figures 1 an d 2 ).

H owever, Ppo5 gene transcripts in event H50 were slightly higher in greenhouse samples than in tubers
from the field. This finding may be due to the fact that the H50 tubers from the greenhouse were not fully mature; some lingering activity of Ppo genes other than Ppo5 , which are only expressed in immature tubers, may have caused residual transcript levels.

Transcript levels for the PhL gene were partially reduced in the tubers of most events ( Figure 3 ) , except F10, F37, J78, and G11

. This reduced PhL was intentional, and is linked to reduced amounts of glucose
and fructose

. It was expected that J78 and G11 would not have reduced transcripts of PhL , yet F10 and F37 were expected to show silencing of that gene. R1 transcripts were partially reduced (“whispered”) in tubers of most lines to help limit the degradation of starch into sugars ( Figure 4 ). Results varied between greenhouse and field grown tubers, yet the overall conclusion was that some silencing of R1
occurred in most events.

Events J78 and G11 lack a functional PhL/R1 promoter silencing cassette and, consequently, display no reductions in transcript levels for the P h L and R1 genes ( Figures 3 and 4 )

. The lack of silencing of PhL in F10 and F37, along with slight reductions of R1 in most events, show that generally promoter silencing of these genes did not down

- regulate genes as much as expected.J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 4 Figure 1

. Expression of the Asn1 G ene in T ubers of Events and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown ( A ) and field

- grown ( B ) tuber tissues of events and controls and hybridized with the Asn1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the Asn1 transcript is 2.0

- kb

. The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed conventional varieties. Individual event samples are labeled above each lane. The events E67 and G9 a re not considered for deregulation.

A. Greenhouse

- grown tuber

B. Field

- grown tubers

EC E12 E24 FC F10 F37 GC G9 G11 HC H37 H50 JC J3 J55 J78

EC= control Russet Burbank, FC = control Ranger Russet, JC = control Atlantic, GC = control G , HC = control H , M = RNA markerJ.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 5 Figure 2

. Expression of the Ppo5 G ene in T ubers of Events and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown ( A ) and field

- grown ( B ) tuber tissues of events and controls and hybridized with the Ppo5 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the Ppo5 transcript is 1.95

- kb (see Genbank Accession
U22921 ). The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed conventional vari eties. Individual event samples are labeled above each lane. The events E67 and G9 are not considered for deregulation.

A. Greenhouse

- grown tubers

B. Field grown

- tubers

EC E12 E24 FC F10 F37 GC G9 G11 HC H37 H50 JC J3 J55 J78

EC= control Russet Burbank, FC = control Ranger Russet, JC = control Atlantic, GC = control G , HC = control H , M = RNA marker

18s rRNA Total RNA Ppo5J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 6 Figure 3

. Expression of the PhL G ene in T ubers of Events and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown ( A ) and field

- grown ( B ) tuber tissues of events and controls and hybridized with the PhL probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the PhL transcript is 4.9

- kb (see Genbank Accession X52385 ). The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed conventiona l varieties. Individual event samples are labeled above each lane. The events E67 and G9 are not considered for deregulation.

A. Greenhouse

- grown tubers

B. Field grown

- tubers

E C E12 E24 FC F10 F37 GC G9 G11 HC H37 H50 JC J3 J55 J78

EC= control Russet Burbank, FC = control Ranger Russet, JC = control Atlantic, GC = control G , HC = control H , M = RNA markerJ.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 7 Figure 4

. Expression of the R1 G ene in T ubers o f Events and Control s

Total RNA (20 μg) w as isolated from greenhouse

- grown ( A ) and field

- grown ( B ) tuber tissues of events and controls and hybridized with the R1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle pa nel). The predicted size of the R1 transcript is 4.8

- kb (see Genbank Accession Y09533 ). The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed conventional v arieties. Individual event samples are labeled above each lane. The events E67 and G9 are not considered for deregulation.

A. Greenhouse

- grown tubers

B. Field grown

- Tubers

E C E12 E24 FC F10 F37 GC G9 G11 HC H37 H50 JC J 3 J55 J78

EC= control Russet Burbank, FC = control Ranger Russet, JC = control Atlantic, GC = control G , HC = control H , M = RNA marker

18s rRNA Total RNA R1J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 8 Target Gene Expression in Stolons

As shown in Figures 5 , 6, 7, and 8 , the Asn1
, Ppo5 , and R1 genes were expressed at low levels in stolons of untransformed plants

. In contrast, high transcript levels in the controls were associated with the P hL

gene.

The transcript levels for the Asn1 gene were lower in stolons from all events , although slight for H37, compared with control stolons from greenhouse

- grown plants ( Figure 5 ). This modification was expected and is not linked to any un desired or unanticipated new phenotype.

Transcript levels of Ppo5 were lowered in events E24, G11, and H50 ( Figure 6 ) . For Ranger Russet and Atlantics, transcript levels in controls and events were at such low levels that silencing was not
detectable.

Expression of the PhL gene was also down

- regulated in the stolons of most events (F10, F37, E12, E24,

J 55, H37, and H50). However, PhL gene expression was unchanged for J78 and G11 stolons, which do not contain a functionally

- active PhL/R1 promoter silencing cassette ( F igure 7 )

. Expression also did not appear to be silenced in stolons of J3.

The amounts of transcript for the R1 gene were slightly reduced (“whispered”) in stolons of most events ( Figure 8 ) . This molecular change contributed to lowering of reducing sugars in tubers

. However, event s J78 and G11 lack the PhL/R1 promoter silencing cassette and, consequently, displayed no reductions in transcript levels for the associated genes in stolons

.J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 9 Figure 5

. Expression of the Asn1 G ene in S tolon T issue of E vents and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the Asn1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the Asn1 transcript is 2.0

- kb. T he lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed conventional varieties. Individual event samples are labeled above each lane. The
events E67
, G9 and H 23 are not considered for deregulation. Event H50 was processed separately with an H control.

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 HC H50

Asn1

Total RNA

18s rRNAJ.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 10 Figure 6

. Expression of the Ppo5 G ene in S tolon T issue of E vents and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the Ppo5 probe (upper panel) and the internal benchmark 18s rRNA prob e (middle panel). The predicted size of the Ppo5 transcript is 1.95

- kb (see Genbank Accession U22921 ). The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed conventional varieties. Individual event samples are labeled above each lane. The events E67
, G9 and H23 are not considered for deregulation

. Event H50 was processed separately with an H control.

Total RNAJ.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 11 Figure 7

. Expression of the PhL G ene in S tolon T issue of E vents and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the PhL probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the PhL transcript is 4.9

- kb (see Genbank Accession X52385). The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed co nventional varieties. Individual event samples are labeled above each lane. The events E67 and G9 are not considered for deregulation. Event H50 was processed separately with an H control.

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 HC H50

PhL

Total RNA

18s rRNAJ.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 12 Figure 8

. Expression of the R1 G ene in S tolon T issue of E vents and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the R1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the R1 transcript is 4.8

- kb (see Genbank Accession Y09533 ). The lower panel shows the amounts of t otal RNA as visualized with ethidium bromide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed conventional varieties. Individual event samples are labeled above each lane. The events E67
, G9 and H23 are not considered for deregulati on

. Event H50 was processed separately with an H control.

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 HC H50

R1

Total RNA

18s rRNAJ.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 13 Transcript Levels in Other Tissues

The Northern expression data for tissues other than tubers and stolons are summarized as below:

(1) In leaf tissues, transcript levels for the Asn1 gene were similar for the events and their untransformed counterparts, except for F10, which contained lower le vels than Ranger Russet (Figure 9) . T ranscript levels for the Ppo5 gene expression were in all cases undetectable under our experimental conditions ( Figure 10 ) . This was expected because the Ppo5 gene would be most active in tubers and expression was not expected in leaves. L evels for the PhL gene were consistently high among the original varieties and their transformed derivatives ( Figure 11 ). Transcript levels for the R1 gene were unaltered in most events when compared to their controls, except for G11, H37, and H50, which contained lower levels than their respective G
and H controls ( Figure 12 ).

(2) In stem tissues, Asn1 gene transcript levels were similar for most events and their controls,

exc ept that F37, E24, J3 , and H50 contained less transcript than the control varieties ( Figure 13 ). Interestingly, transcript levels for the Ppo5 gene were reduced in all events submitted for deregulation, except for G11 ( Figure 1 4 ). PhL gene expression was similar in all events and their controls ( Figure 15 ), and R1 gene expression was reduced in G11 only (Figure 16 )

.
(3) In root tissue s , transcript levels for the Asn1 gene were reduced in six events ( F10, F37, E12, J3, J55, and H50), and remained unchanged f or E24, J78, G11, and H37 ( Figure 17 ) . Transcript levels
for the Ppo5 gene were reduced in F10, F37, E12, H37, and H50 ( Figure 18 ). This indicates that the promoters used to drive silencing are partially functional in underground tissues. In Atlantic
and G varieties, low levels of transcripts were observed in both controls and events, suggesting
that Ppo5 gene activity may be naturally lower in roots of those varieties. No significant reductions in expression levels were observed for the PhL gene, and for the R1 gene F10, F37, and H50 were lower ( Figures 19 and 20 ).

(4) In floral tissue s , Asn1 gene transcript levels were lower for the 8 events (the H variety does not flower, so no data were obtained for H37 and H50) than for the original varieties ( Figure 2 1 ). Transcripts were not detectable for the Ppo5 gene ( Figure 22 ). Expression levels of the PhL gene for all events were not reduced compared with their untransformed controls (Figure 2 3 )

. For
the R1 gene , expression levels in F37 were lower than the Ranger Russet control , but all other
events were similar to controls (Figure 24)

.
It can be concluded that transcript levels of the four targeted genes were occasionally down

- regulated in tissues other than tubers and stolons. In these cases, the reductio ns were partial and were not associated with agronomic differences in the events when compared to untransformed controls in field
trials.J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 14 Target G ene E xpression i n L eaf T issue

Tran script levels in leaf tissues were determined by using greenhouse

- grown plants, assuming that performance in the greenhouse will predict what occurs in the field. Indeed, there is extensive
evidence in the scientific literature ( Anand et al

. 2003; Kuipers et al

. 1992; Mik schofsky et al

. 2011 ) to support that hypothesis

.
In leaf tissues, transcript levels for the Asn1 gene were similar for the events and their untransformed counterparts, except for F10, which contained lower levels than Ranger Russet (Figure 9) . Transcrip t levels for the Ppo5 gene expression were in all cases undetectable under our experimental conditions ( Figure 10 ). This was expected because the Ppo5 gene would be most active in tubers and expression was not expected in leaves. Levels for the PhL gene were consistently high among the original varieties and their transformed derivatives ( Figure 11 ). Transcript levels for the R1 gene were unaltered in most events when compared to their controls, except for G11, H37, and H50, which contained lower levels t han their respective G and H controls ( Figure 12 ).

.J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 15 Figure 9

. Expression of the Asn1 G ene in L eaf T issue of E vents and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the Asn1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the Asn1 transcript is 2.0

- kb. The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed conventional varieties. Individual event samples are labeled above each lane. The events
E67
, G9 and H23 are not considered for deregulation

. Event H50 was processed separately with an H control.

M E C E12 E24 E67 FC F10 F37 JC J 3 J55 J78 GC G9 G11 HC H23 H37 HC H50

EC= control Russet Burbank, FC = control Ranger Russet, JC = control Atlantic, GC = control G , HC = control H, M = RNA markerJ.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 16 Figure 10

. Expre ssion of the Ppo5 G ene in L eaf T issue of E vents and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the Ppo5 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the Ppo5 transcript is 1.95

- kb (see Genbank Accession U22921 ). The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed conventional varieties. Individual event samples are labeled above each lane. The events E67
, G9 and H23 are not considered for deregulation

. Event H50 was processed separately with an H control.

EC= control Russet Burbank, FC = control Ranger Russet , JC = control Atlantic, GC = control G , HC =
control H, M = RNA markerJ.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 17 Figure 11

. Expression of the PhL G ene in L eaf T issue of E vents and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the PhL probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the PhL transcript is 4.9

- kb (see Genbank Accession X52385). The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed conventional varieties. Individual event samples are labeled above each lane. The events E67

, G9 , and H23 are not considered for deregulation. Event H50 was processed separately with an H control.

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 M HC H50



### 18S rRNA Total RNA PhLJ.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 18 Figure 12

. Expression of the R1 G ene in L eaf T issue of E vents and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the R1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the R1 transcript is 4.8

- kb (see Genbank Accession Y09533 ). The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed conventional varieties. Individual event samples are labeled above each lane. The events E67
, G9 a nd H23 are not considered for deregulation

. E v ent H 5 0 was p ro c esse d separately with an H control.

EC= control Russet Burbank, FC = control Ranger Russet, JC = control Atlantic, GC = control G , HC =
control H, M = RNA marker

M EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 

### HC H50

18S rRNA Total RNA R1J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 19 Target G ene E xpression in S tems

In stem tissues, Asn1 gene transcript levels were similar for most events and their controls, except that F37, E24, J3, and H50 contained less transcript than the control varieties ( Figure 13 ). Interestingly, transcript levels for the Ppo5 gene were reduced in all events submi tted for deregulation, except for G11 ( Figure 14 ). PhL gene expression was similar in all events and their controls ( Figure 15 ), and R1 gene expression was reduced in G11 only (Figure 16 )

.

Figure 13

. Expression of the Asn1 G ene in S tem T issue of E vents and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the Asn1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the Asn1 transcript is 2.0

- kb. The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed conventional varieties. Individual event samples are labeled above each lane. The events
E67
, G9 and H23 are not considered for deregulation

. Event H50 was processed separately with an H control.

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 HC H50



### 18S rRNA Total RNA Asn1J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 20 Figure 1 4

. Expression of the Ppo5 G ene in S tem T issue of E vents and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the Ppo5 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel) . The predicted size of the Ppo5 transcript is 1.95

- kb (see Genbank Accession U22921 ). The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed conventional va rieties. Individual event samples are labeled above each lane. The events E67
, G9 and H23 are not considered for deregulation

. Event H50 was processed separately with an H control.

EC= control Russet Burbank, FC = control Ranger Russet, JC = control Atlantic, GC = control G , HC =
control H, M = RNA marker

M EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 HC H50

18S rRNA Total RNA Ppo5J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 21 Figure 1 5

. Expression of the PhL G ene in S tem T issue of E vents and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the PhL probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the PhL transcript is 4.9

- kb (see Genbank Accession X52385). The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed conventional varieties. Individual event samples are labeled above each lan e. The events E67
, G9 and H23 are not considered for deregulation

. Event H50 was processed separately with an H control.

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 M 

### HC H50

18S rRNA Total RNA PhLJ.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 22 Figure 1 6

. Expression of the R1 G ene in S tem T issue of E vents and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the R1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the R1 transcript is 4.8

- kb (see Genbank Accession Y09533 ). The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed con ventional varieties. Individual event samples are labeled above each lane. The events E67
, G9 and H23 are not considered for deregulation

. Event H50 was processed separately with an H control.

EC= control Russet Burbank, FC = control Ranger Russet, JC = control Atlantic, GC = control G , HC =
control H, M = RNA marker

M EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 

### HC H50

18S rRNA Total RNA R1J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 23 Target G ene E xpression in R oots

In root tissue s, transcript levels for the Asn1 gene were reduced in six events (F10, F37, E12, J3, J55, and H50), and remained unchanged for E24, J78, G11, and H37 ( Figure 17 ) . Transcript levels for the Ppo5 gene were reduced in F10, F37, E12, H37, and H50 ( Figure 18 ) . This indicates that the promoters used to drive silencing are partially functional in underground tissues. In Atlantic and G varieties, low levels of transcripts were observed in both controls and events, suggesting that Ppo5 gene activity may be

natura lly lower in roots of those varieties. No significant reductions in expression levels were observed
for the PhL gene, and for the R1 gene F10, F37, and H50 were lower ( Figures 19 and 20 ).

Figure 17

. Expression of the Asn1 G ene in R oot T issue of E vents a nd Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the Asn1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the Asn1 transcript is 2.0

- kb. The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed conventional varieties. Individu al event samples are labeled above each lane. The events
E67
, G9 and H23 are not considered for deregulation

. Event H50 was processed separately with an H control.

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H 37 HC H50

Asn1

Total RNA

18S rRNAJ.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 24 Figure 18

. Expression of the Ppo5 G ene in R oot T issue of E vents and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the Ppo5 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the Ppo5 transcript is 1.95

- kb (see Genbank Accession U22921 ). The lower panel shows the amounts of total RNA as visualized with ethidium brom ide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed conventional varieties. Individual event samples are labeled above each lane. The events E67
, G9 and H23 are not considered for deregulation

. Event H50 was processed separately with an H control.

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 HC H50

Ppo5

Total RNA

18S rRNAJ.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 25 Figure 19

. Expression of the PhL G ene in R oot T issue of E vents and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the PhL probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the PhL transcript is 4. 9

- kb (see Genbank Accession X52385). The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed conventional varieties. Individual event samples are labeled abov e each lane. The events E67
, G9 and H23 are not considered for deregulation

. Event H50 was processed separately with an H control.

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 HC H50

PhL

Total RNA

18S rRNAJ.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 26 Figure 20

. Expression of the R1 G ene in R oot T issue of E vents and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the R1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the R1 transcript is 4.8

- kb (see Genbank Accession Y09533 ). The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB) . EC, FC, JC, GC and HC are control samples from the untransformed conventional varieties. Individual event samples are labeled above each lane. The events E67
, G9 and H23 are not considered for deregulation

. Event H50 was processed separately with an H control.

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 HC H50

R1

Total RNA

18S rRNAJ.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 27 Target Gene E xpression in Floral T issue

In floral tissues, Asn1 gene transcript levels were lower for the 8 events (the H variety does not flower, so no data were obtained for H37 and H50) than for the original varieties ( Figure 21 ). Transcripts were not detectable for the Ppo5 gene ( Figure 22 ). Expression levels of the PhL gene for all events were not reduced compared with their untransformed controls (Figure 2 3 ) . For the R1 gene, expression levels in F37 were lower than the Ranger Russet control, but all other events were similar to controls (Figure 24)

.

Figure 21

. Expression of the Asn1 G ene in F loral T issue of E vents and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the Asn1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the Asn1 transcript is 2.0

- kb. The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB) . EC, FC, JC, and GC are control samples from the untransformed conventional varieties. Individual event sam ples are labeled above each lane. The events E67 and G9 are not considered for deregulation

.

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11

18S rRNA Total RNA Asn1J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 28 Figure 22

. Expression of the Ppo5 G ene in F loral T issue of E vents and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the Ppo5 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the Ppo5 transcript is 1.95

- kb (see Genbank Accession U22921 ). The lower panel shows the amo unts of total RNA as visualized with ethidium bromide (EB)

. EC, FC, JC, and GC are control samples from the untransformed co nventional varieties. Individual event samples are labeled above each lane. The events E67 and G9 are not considered for deregulation

.

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9

18S rRNA Total RNA Ppo5J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 29 Figure 23

. Expression of the PhL G ene in F loral T issue of E vents and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the PhL probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the PhL transcript is 4.9

- kb (see Genbank Accession X52385). The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB)

. EC, FC, JC, and GC are control samples from the untransformed conventional varieties. Individual event samples are labeled above each lane. The events E67 and G9 are not considered for deregulation

.

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11

18S rRNA Total RNA PhLJ.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 30 Figure 24

. Expression of the R1 G ene in F loral T issue of E vents and Controls

Total RNA (20 μg) w as isolated from greenhouse

- grown tissues of events and controls and hybridized with the R1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The predicted size of the R1 transcript is 4.8

- kb (see Genbank Accession Y09533 ). The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB)

. EC, FC, JC, and GC are control samples from the untransformed conventional varieties. Individual event samples are labeled above each lane. The events E67 and G9 are not considered for deregulation

.

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11

18S rRNA Total RNA R1J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 31 SUMMARY

It was concluded that transcript levels of the four targeted genes were mostly down

- regulated in tubers of the events that carry at least one intact DNA insert , as would be expected from using tuber/stolon

specific promoters

. For some events, E24 and H50, all four genes were also down

- regulated in stolons.
The two events J78 and G1 1, which contain a truncated DNA insert with a functionally active Asn1 / Ppo5 silencing cassette, produced tubers that contained reduced transcript levels for the Asn1 and Ppo5 genes only , since they did not contain the PhL/R1 silencing cassette

. Overall, more down

- regulation of the four targeted genes occ urred in tubers and stolons than in the roots, stems, leaves, or flowers ( Table

1 ).

The selected events were more strongly affected in the expression levels of the Asn1 and Ppo5 genes than in those of the R1 and PhL genes. This result coincided with our specific interest in (1) preventing
the formation of Ppo protein and free ASN to the greatest extent possible , and (2) only partially blocking the conversion of starch into glucose and fructose.

There were occasional changes in transcript levels in tissues other than tubers and stolons

, demonstrating some leakiness of the tuber/stolon

- specific promoter ( Table 1 ). Such event

- specific variations may have come about because of position effects. For instance, i t is possible that the driving promoters within the DNA insert are enhanced in activity by genetic elements in adjacent plant DNA. An alternative explanation is that small RNAs produced in tubers through expression of the silencing cassettes migrate into o ther tissues, especially roots and stems ( Molnar et al. 2010 ). In most cases of altered expression levels in tissues other than tubers and stolons, the differences were found to be minor. Unanticipated reductions in gene expression in non

- tuber tissues we re not associated with agronomic differences in the field and are, therefore, not expected to have any impact on the safety of the plants or their pest potential.J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 32 Table 1. Summary of Down

- Regulated Genes in Different Tissues

Event Tubers 1 Stolons 1 Roots 1 Stems 1 Leaves 1 Flowers 1, 2

F10 A P R A L R A P R P A A F37 A P R A L R A P R A P A R
E12 A P L R A L R A P P A
E24 A P L R A P L R A P A
J3 A P L R A R A A P A
J55 A P L R A L R A P A
J78 A P A P A
G11 A P A P R R A
H37 3 A P L R L R P P R
H50 3 A P L R A P L R A P R A P R

1 A = Asn1 , P = Ppo 5, L = PhL , R = R1 . Letters in table indicate down

- regulated gene expression by tissue.

2 The partially down

- regulated Asn1 gene expression in flowers might alter the amino acid composition of the event flowers. Such effects will be limited to a reduction in ASN and an in crease in GLN. Since ASN and GLN are similar

non

- essential a mino acids, changes in the levels of these compounds is not expected to affect the quality of petal, nectar, and pollen as feed for insects or other organisms.

3 Events derived f rom variety H do no t produce flowers.J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 33 MATERIALS AND METHODS

Plant material. Gene expression levels were determined by carrying out RNA gel blot analyses on tubers, stolons, roots, stems, leaves, and flowers of greenhouse

- grown plants of all events and controls.
G eneratio n 2 (G2) tubers from the field (Othello

, 2010 for Ranger Russet, Russet Burbank and Atlantic, and Rhinelander, 2010 for the G and H variet ies ) were subjected to this analysis as well. Three replications per event were used in the analysis.

RNA Isolation. RNA was extracted from 1 g of tuber, stolon and root tissue using Plant RNA reagent (Invitrogen, Carlsbad, California), whereas Trizol reagent (Invitrogen) was used to extract RNA from leaves, flowers and stems. The concentration of isolated RNA was measur ed spectrophotometrically at 260 nm, and RNA quality was confirmed by running a sample on a 1% agarose gel with 1X MOPS buffer (200 mM MOPS, 50 mM NaOAc , 20 mM EDTA , pH7.0) for 30

- 60 min at 90 volts.

RNA transfer. 20

- 25  g of RNA was denatured in RNA loading dye (Sigma) for 10

- min at 65 0 C followed by incubation on ice for 5

- min. Denatured RNA was loaded onto an RNA gel containing 1% agarose, 1X MOPS, 0.1

- 0.25  g/ml ethidium bromide and 2% formaldehyde (5.5 ml 36.5% stock in 100 ml solution). The gel w as run at 80

- 85 volts for 2

- 3 hrs and then photographed using the gel documentation system from Alpha Innotech (Santa Clara, California). The gel was soaked twice in 10 X SSC for 15

- min to remove the formaldehyde. RNA was transferred from the gel to a posit ively charged nylon membrane (Roche, Indianapolis) by capillary blotting with 10X SSC for 16

- 18 hrs, and the transferred RNA was stabilized onto the filter by UV cross

- linking (UVP, Upland, California). The RNA

- containing membrane was stored

at 4 0 C.

Prepar ation of DIG labeled Probe. A PCR based method was used to prepare DIG labeled probes for four target transcripts and an internal control of 18s rRNA. The primers used for PCR amplification and the length s of probes are listed in Table 2 . A typical 50 μl l abeling reaction consisted of 5 μl HotMaster Taq Buffer (Fisher BioReagents), 2

- 5 μl of 10  M forward primer, 2

- 5  l of 10 μM reverse primer, 5μl
DIG

- labeled dNTP (Roche, Indianapolis), 5

- 30 ng plasmid template, 0.50

- 0.75 μl HotMaster Taq polymerase, and dH 2 O for a total volume of 50 μl. PCR conditions were specific for each DIG

- labeled probe. The DIG

- labeled probe was checked on 1% agarose gel and always ran slower than the control PCR product. The probe was denatured before use by incubating for 5 min at 100 0 C and then transferring to ice.

Hybridization. Nylon membranes containing transferred RNA were pre

- hybridized in 40 ml pre

- warmed DIG Easy Hybridization solution (Roche, Indianapolis) for at least 1

- 4 hrs at 20

- 25 rpm in a hybridization

oven set at 4 2 O C (Amerex Instruments). The hybridization solution was replaced by a mix of 40 ml fresh

pre

- warmed hybridization solution and 25

- 50μl of denatured DIG

- labeled probe, and the membrane was
incubated in this mix for 3

- 16 hrs at 42 O C. The hybridization sol ution can be store at

- 20 0 C and reused. The reused hybridization solution was heated at 68 0 C for 10 minutes before use.J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 34 Table 2

. Primers and P robes U sed for E xpression A nalysis of P otato G enes Probe

name Hybridization

target Primer sequences Probe size (bp)

ASN1 Asn1 transcript F: GGTTGATGACTGATGTCCCCTTTG
R: TAGTTAGCTCCTTATTGTGAGCTC 1115

PPO 1 Ppo5 transcript F: ATCTTCCACTCCTAAGCCCTCTCAAC R: CCGCCAAAGAACATCCGAGG 864

PHL PhL transcript F: CAATTCCAGATTCATCCATTTCAC R: CATCCCCAGGGTAGAGTATGTAAC 968

R1 R1 transcript F: ACCAGGGATTCCTAACCTCAAC R: CAGGTATATCACTCTTTGTTAC 852

18s 18s rRNA F: GCATTTGCCAAGGATGTTTT
R: GTACAAAGGGCAGGGACGTA 694

1 The PPO probe was derived from a cDNA of the Ppo5 gene of S. tuberosum , which is highly expressed in tubers. The probe also hybridized with Ppo genes expressed in other tissues. The sequence in pSIM1278 used to silence the Ppo5 gene consists of the untranslated trailer of a Ppo gene from S. verrucosum

.

Detection. The hybridization solution was removed and replaced by 100 ml washing solution I (2X SSC/0.1% SDS). Nylon membranes were washed twice in washing solution I for 10 min at RT in the hybridization oven at 25

- 30 rpm. This low stringency buffer was poured off and preheated high stringency washing solution II (0.5X SSC/ 0.1% SDS) was immediately added. The membrane was washed twice in washing solution II at 60 0 C for 20 min each at 25

- 30 rpm. This was followed by a wash for 20 min with washing solution III (0.1X SSC/ 0.1% SDS) at 65 0 C and a brief rinse with 2X SSC to remove SDS. The membrane was rinsed with 100 ml 1X DIG Washing Solution (Roche, Indianapolis) in a tray at room temperature for 2 min. and incubated in 1X Blocking solution (Roche, Indianapolis) for 3 0 min. to 3 hrs on a shaker. The blocked membrane was incubated with DIG antibody solution (1:10,000 dilution of
Anti

- DIG

- alkaline phosphate conjugate with 1X Blocking solution, Roche, Indianapolis) for 30 min on a shaker. The membrane was washed twice (15 min each) with 1X DIG Washing Solution and equilibrated
with 1X detection buffer. The detection reaction was carried out with 2 ml CDP

- Star solution (1:100
diluted stock of CDP

- Star with 1X detection buffer) for 5 min. The membrane was wrapped in a plasti c film and exposed to Z

- ray film in the dark. Depending on the experiment multiple exposures were taken from 30 sec to 10 min). The films were developed with Konica SRX

- 101A X

- ray film developer. The x

- ray films were scanned to obtain electronic images.J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 35 CONCLUSION S

The targeted four genes ( Asn1, Ppo5, PhL, and R1 ) are partially silenced in tubers of most events, except for J78 and G11 which do not contain the cassette for silencing the Ph L /R1 genes ( Table 1 ). In addition
the PhL gene did not appear down

- regulated in tubers of the Ranger Russet e vents, F10 or F37. The
Asn1 and Ppo5 gene s are down

- regulated in stolons, roots and stems for some of the events. The Asn1 gene was down regulated in flowers of all events with the exception of the H variety that does not produce flowers. These results indicate that the promoters used for gene silencing are not entirely
tissue

- specific.

Considering the potato events in this study, expression of the targeted genes would provide evidence that the desired pheno type with low asparagine, reduced black spot bruise , and lowered reducing sugars had been achieved. It could provide information relevant to the plant pest risk assessment, for example, all Innate TM potatoes in the study had reduced or similar expression t o the controls. Thus, the altered expression is unlikely to affect weediness or other plant pest characteristics. Also, with these events, the expression studies pertain to endogenous proteins rather than new proteins that could require a safety evaluati on, such as those associated with herbicide tolerance or insect resistance. The altered gene expression levels did not affect the agronomic characteristics or tuber composition of the event compared to the conventional untransformed variety.

Supporting data for th ese conclusion s are available in Appendix 6. Field Performance and Tuber
Evaluations and Appendix 9

. Compositional Analyses

.
Event Ranger Russet F10: The targeted genes are at least partially silenced in tubers , except for PhL, and
all but Ppo5 are silenced in stolons . The Asn1
, Ppo5 , and R1 genes are down

- regulated in roots

, the Ppo5 gene in stems
, and the Asn1 gene in leaves and flowers ( Table 1 ).

Event Ranger Russet F37: The targeted four genes are at least partially silenced in tubers , exce pt for PhL, , and all but Ppo5 are silenced in stolons . Some of these are also down

- regulated in expression, at least partially, in roots ( Asn1
, Ppo5 , and R1 ), stems ( Asn1 and Ppo5 ), and flowers ( Asn1 and R1 ) ( Table

1 ).

Event Russet Burbank E12: The target ed four genes are at least partially silenced in tubers , and all but Ppo5 are silenced in stolons

. Some of these are also down

- regulated in expression, at least partially, in roots ( Asn1 and Ppo5 ), stems ( Ppo5 ), and flowers ( Asn1 ) ( Table 1 ).

Event Russet Burbank E24: The targeted four genes are at least partially silenced in tubers and stolons.
Some of these are also down

- regulated in expression, at least partially, in stems ( Asn1 and Ppo5 ) and flowers ( Asn1 ) ( Table 1 ).

Event Atlantic J3: The tar geted four genes are at least partiall y silenced in tubers, and Asn1 and R1 are silenced in stolons . Some of these are also down

- regulated in expression, at least partially, in roots ( Asn1 ), stems ( Asn1 and Ppo5 ), and flowers ( Asn1 ) ( Table 1 ).J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 36

Event Atla ntic J55: The targeted four genes are at least partially silenced in tubers, and all but Ppo5 are silenced in stolons . Some of these are also down

- regulated in expression, at least partially, in roots ( Asn1 ), stems ( Ppo5 ), and flowers ( Asn1 ) ( Table 1 ).

Ev ent Atlantic J78: The Asn1 and Ppo5 genes are silenced in tubers, the Asn1 gene is silenced in stolons,
the Ppo5 gene is silenced in stems, and the Asn1 gene is silenced in flowers ( Table 1 ). Silencing was not
observed for the PhL

/R1 genes, as expected

, since J78 does not contain the cassette for silencing the
PhL /R1 genes.

Event G11: The Asn1 and Ppo5 genes are silenced in tubers and stolons; the R1 gene is partially silenced
in leaves and stems ; and the Asn1 gene is silenced also in flowers ( Table 1 ). Silencing in tubers and
stolons was not observed for the PhL /R1 genes, as expected s ince G11 does not contain the cassette for silencing the PhL

/R1 genes. The lower expression of R1 in leaves and stems was not expected. Such unanticipated reductions in g ene expression in non

- tuber tissues were not associated with agronomic differences in the field and are, therefore, not expected to have any impact on the safety of G11 plants or their pest potential.

Event H37: The targeted four genes are at least partial ly silenced in tubers , and PhL and R1 are silenced
in stolons . Some of these are also down

- regulated in expression, at least partially, in roots ( Ppo5 ), stems ( Ppo5 ), and R1 is silenced in leaves ( Table 1 ).

Event H50: The targeted four genes are at least partially silenced in tubers and stolons. Some of these
are also down

- regulated in expression, at least partially, in roots ( Asn1
, Ppo5
, and R1 ), stems ( Asn1 and Ppo5 ), and R1 is silenced in leaves ( Table 1 ).J.R. Simplot Company Efficacy and Tissue

- Specificity of Gene Silencing 37 REFERENCES

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