# US 2017/0254801 A1 · Method for identifying substances which prime a defense response

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- Number: US 2017/0254801 A1
- Document ID: US20170254801A1
- Status: Published application
- Kind: A1
- Application number: 15/506,599
- Filed: 2015-08-27
- Published: 2017-09-07
- Earliest claimed: 2014-08-27
- Assignees: Rheinisch WestfäLische Technische Hochschule Aachen (Rwth)
- Applicants: Rheinisch-WestfäLische Technische Hochschule Aachen (Rwth)
- Inventors: Britta SCHILLHEIM; Jochen BÜCHS; Uwe CONRATH; Jana SCHILLING
- Web page: https://patentgrep.com/patent/US20170254801A1

## CPC

- G01N 33/5097: {involving plant cells (immunoassays of plant cells G01N33/56961)}
- G01N 33/5038: {involving detection of metabolites per se}
- G01N 2500/10: involving cells
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention relates to a method for identifying substances which prime cells for a stress response by determining the respiration activity of the cells treated with a candidate substance in comparison to cells not treated with the candidate substance.

## Claims

### Claim 1

A method for identifying substances which prime cells for a stress response comprising the steps of:
inoculating cells in a suitable culture medium and culturing them in said medium;
adding one or more candidate substances to the culture medium thereby treating the cells with said candidate substance; and
measuring the respiration activity in the cells treated with the candidate substance and in control cells not treated with the candidate substance, wherein an increase in the respiration activity in the cells treated with the candidate substance compared to the control cells indicates that the candidate substance primes the cells for a stress response.

### Claim 2 (depends on 1)

The method according to claim 1, wherein measuring the respiration activity comprises measuring the oxygen transfer rate.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the cells are plant or mammalian cells.

### Claim 4

A method for identifying substances which prime plant cells for a stress response comprising the steps of:
inoculating plant cells in a suitable culture medium and culturing them in said medium;
adding one or more candidate substances to the culture medium thereby treating the plant cells with said candidate substance; and
measuring the oxygen transfer rate in the cells treated with the candidate substance and in control cells not treated with the candidate substance,
wherein an increase in the oxygen transfer rate in the cells treated with the candidate substance
compared to the control cells indicates that the candidate substance primes the plant cells for a stress response.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the candidate substance is added 24 to 120 hours after inoculation.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the cells are cultured in suspension.

### Claim 7 (depends on 2)

The method according to claim 2, wherein the candidate substance is added when the oxygen transfer rate is about twice the oxygen transfer rate at the time of inoculation.

### Claim 8 (depends on 1)

The method according to claim 1, further comprising, after the addition of the candidate substance, subjecting the cells to a compound eliciting a stress response.

### Claim 9 (depends on 8)

The method according to claim 8, wherein the cells are subjected to the compound eliciting a stress response 6 to 48 hours after the addition of the candidate substance.

### Claim 10 (depends on 2)

The method according to claim 2, wherein a difference in respiration activity between cells treated with the candidate substance and control cells is quantified using the formula I:
oxygen


consumption


(
t
1
-
t
4
)

[
%
]
=
(
∫
t


1
t


4

OTR
+
priming


compound
∫
t


1
t


4

OTR
no


additives
-
1
)
·
100
wherein ∫t1t4 OTR+ priming compound is the area under the curve for cells treated with the candidate substance, ∫t1t4 OTRno additives is the area under the curve for the untreated control cells, t1 is the point in time at which the candidate substance is added and t4 is the point in time after addition of the candidate substance at which the oxygen transfer rate of the cells treated with the candidate substance is the same as the oxygen transfer rate of the untreated cells.

### Claim 11 (depends on 8)

The method according to claim 8, wherein a difference in respiration activity between cells treated with the candidate substance and control cells is quantified using the formula II:
oxygen


consumption


(
t
1
-
t
2
)

[
%
]
=
(
∫
t


1
t


2

OTR
+
priming


compound
∫
t


1
t


2

OTR
no


additives
-
1
)
·
100
wherein ∫t1t2 OTR+priming compound is the area under the curve for cells treated with the candidate substance, ∫t1t2 OTRno additives is the area under the curve for the untreated control cells, t1 is the point in time at which the candidate substance is added and t2 is the point in time at which the cells are subjected to a compound eliciting a stress response.

### Claim 12 (depends on 8)

The method according to claim 8, wherein a relative increase of the respiration activity upon subjecting the cells to the compound eliciting a stress response in cells treated with the candidate substance in comparison to the control cells is quantified using the formula III:
Relative


Δ


OTR
max

[
%
]
+
(
Δ


OTR
max

1
Δ


OTR
max

2
-
1
)
·
100
wherein ΔOTRmax1 is the difference of the oxygen transfer rate between the peak oxygen transfer rate after adding the compound eliciting a stress response and the oxygen transfer rate at the time at which the compound eliciting a stress response is added in cells treated with the candidate substance and a compound eliciting a stress response and ΔOTRmax2 is the difference of the oxygen transfer rate between the peak oxygen transfer rate after adding the compound eliciting a stress response and the oxygen transfer rate at the time at which the compound eliciting a stress response is added in the control cells only treated with the compound eliciting a stress response.

### Claim 13 (depends on 8)

The method according to claim 8, wherein a difference in respiration activity between cells treated with the candidate substance and control cells is quantified using the formula IV:
oxygen


consumption


(
t
2
-
t
3
)

[
%
]
=
(
∫
t


2
t


3

OTR
+
priming


compound
/
+
elicitor
∫
t


1
t


3

OTR
+
elicitor
-
1
)
·
100
wherein ∫t2t3 OTR+priming compount/+elicitor is the area under the curve for cells treated with the candidate substance and subjected to the compound eliciting a stress response, ∫t2t3 OTR+elicitor is the area under the curve for control cells only subjected to the compound eliciting a stress response, t2 is the point in time at which the cells are subjected to the compound eliciting a stress response and t3 is the point in time 12 to 48 hours after subjecting the cells to the compound eliciting a stress response.

### Claim 14 (depends on 8)

The method according to any of claim 8, wherein a difference in respiration activity between cells treated with the candidate substance and control cells is quantified by calculating a linear regression of the oxygen transfer rate curve for the time before adding the candidate substance and an exponential regression of the oxygen transfer rate curve for the time after adding the candidate substance and determining the tangent slope of the exponential regression at the interception of the linear and exponential regression and/or by calculating the ratio of the slope of the tangent of the exponential regression to the slope of the linear regression according to the formula VI:
Ratio

[
-
]
=
a
·
b
·
e
a

(
c
-
t
)
m

### Claim 15 (depends on 1)

The method according to claim 1, wherein the cells are from Petroselinum crispum, Triticum aestivum, Glycine max, Solatium tuberosum, Oryza sativa or Zea mays.

### Claim 16

(canceled)