JAZ Genes Levels after Infection of Parasitic Root-Knot Nematode in Arabidopsis

Ashley Smith, Mentor: Heather Marella Bridgewater State University, Department of Biological Sciences, Bridgewater, MA 02325

Root-knot nematodes are a major problem in the United States and around the world. They damage billions of dollars’ worth of crops every year. So I want to look at how the root-knot nematode affects the JAZ

gene family in the plant Arabidopsis. These genes are turned on by the plant hormone, Jasmonic acid, and are used to defend the plant from

predators or pathogens. There are 12 JAZ genes in Arabidopsis but we do not know if they each have distinct roles in Jasmonic acid signaling. I examined the levels of the Arabidopsis JAZ genes in the presence and

absence of nematodes. Arabidopsis is a plant that is quick growing with its whole genome sequenced making it easy to use for the genetic tests

that I performed on the JAZ gene family. I grew Arabidopsis in sterile petri dishes for about two weeks in an incubator then added the juvenile nematodes. After one day and five days, I extracted RNA from the roots. I

then turned this RNA into complimentary DNA (cDNA) by reverse transcription and used it for real time Polymerase Chain Reaction (qPCR) to look at how the nematodes affect the levels of the JAZ genes. I hope to identify which of the 12 JAZ genes family members in the Arabidopsis are

altered during nematode parasitism.

Abstract

Background Results

Acknowledgements I would like to thank the Office of Undergraduate Research for funding supplies for my experiments. I especially would like to thank Heather Marella for helping me learn the laboratory techniques and assisting me in interpreting my results.

References

Methods

Plants are sedentary organisms that cannot defend themselves in the typical sense like animals. Plants use specialized hormones to defend themselves from the living organisms that invade or injury their body. One of these hormones is Jasmonic acid. There are 12 different genes that activate Jasmonic acid signaling, called the JAZ gene family, and each has their own role that aid in the protection of the plant. Jasmonic acid is important when parasitic nematodes infect plant roots. This was discovered when cell death occurred and Jasmonic acid was activated in the model plant, Arabidopsis thaliana (Benedito 2015). Parasitic nematodes, like the root-knot nematode, Meloidogyne incognita, infect a plant near the root tip. The J2s or the infective stage of the root-knot nematode damages cells by taking their stylet and damaging the cell wall and also breaking it down with enzymes (Jones et. al 2013). These nematodes bury themselves into the root tissue and feed from redifferentiated cells, called giant cells, near the vascular tissue of the plant. Unfortunately this depletes the plant of its nutrients which will negatively impact growth. This is very prevalent in agricultural settings and is spread worldwide to almost every vascular plant (Jones et. al 2013). I am interested to see how the levels of these 12 JAZ genes change in the presence of the root-knot nematode and what their function might be in protecting the plant. Using real time PCR, I am measuring the levels of all 12 JAZ genes in the roots of nematode infected and also uninfected roots to identify those with altered levels of expression.

Plated sterile Arabidopsis seeds

on 4 plates and allowed to grow for

two weeks.

After two weeks Juvenile Root-

Knot Nematodes (RKN) were

added to two plates; the other two plates were controls with no

RKN.

RNA was then extracted from the roots of the seedlings from the four different plates. Two plates, one control and one RKN plate, were extracted after one day of infection and the other pair (a control

and a RKN plate) were extracted after five days of infection.

RNA was then turned into cDNA

by reverse transcription. This

cDNA was used for PCR

expression testing.

Gel electrophoresi

s was run to visualize the PCR products and that the

cDNA process ran correctly.

cDNA from each plate was then

amplified with JAZ gene primers to

test the levels and presence of each

gene through qPCR.

DNA

Ladd

er

Actin

– D

ay 1

, No

J2’s

Ac

tin –

Day

1, J

2’s

Actin

– D

ay 5

, No

J2’s

Ac

tin –

Day

5, J

2’s

PP2A

A3 –

Day

1, N

o J2

’s PP

2AA3

– D

ay 1

, J2’

s PP

2AA3

– D

ay 5

, No

J2’s

PP

2AA3

– D

ay 5

, J2’

s Li

pase

– D

ay 1

, No

J2’s

Li

pase

– D

ay 1

, J2’

s Li

pase

– D

ay 5

, No

J2’s

Li

pase

– D

ay 5

, J2’

s

Figure 1: Gel Electrophoresis to Test Presence of cDNA with Different Primer Sets.Lane 1 contains a DNA ladder to measure DNA size. Lanes 3 through 6 contain cDNA with primers for the Actin gene. Lanes 8 through 11 contain cDNA with primers for PP2AA3. Then lanes 13 through 16 contain cDNA with primers for Lipase. All of these are genes that are unaffected by the presence of a nematode and show whether or not the cDNA synthesis worked.

Figure 2: Melt Curve for JAZ3 qPCR. One curve is present showing only one target was amplified.

Figure 4: Levels of Gene Expression in Specific JAZ Genes by qPCR.All of these genes depicted in the graphs, JAZ 1 (red), JAZ 12 (bright orange), JAZ 9 (maroon), JAZ 6 (dark orange), and JAZ 10 (blue) (depicted in that order from top to bottom), have interesting trends compared to the null hypothesis. JAZ 6, 9, and 12 all increased dramatically on day 5 with no root-knot nematodes while JAZ 1 and 10 decreased as compared to the 1 day no nematode control. Additionally, all five genes show strong induction of expression with the addition of nematodes at day 5.

Conclusions• The expression of JAZ 1, 6, 9, 10, and 12 change over the course of root development and increase response to nematode infection.

More qPCR will be performed to confirm the patterns observed in this initial experiment.• The expression of JAZ 2, 3, 5, 7 and 11 did not change during the scope of this experiment and will not be focused on in the future. • I will also repeat JAZ 4 because of technical issues with the qPCR for this gene. • The cDNA for day 1 with J2’s was not successfully synthesized so I will be repeating the plant growth and RKN infection to generate

more biological replicates next semester.

Figure 3: Amplification Plots for JAZ9 (A) and JAZ12 (B) qPCR. The detection of SYBR Green fluorescence during the qPCR is plotted according to the PCR cycle at which its level crossed the background level of fluorescence. The 1 day no RKN samples are the red lines, the 5 day no RKN are the green lines, and the 5 day with RKN are the yellow lines. JAZ9 is not expressed in the 1 day no RKN samples.

A

B

Benedito, V. ed. (2015). Jasmonates: Defense and More. The Plant Cell: 1 – 13.

Jones JT, Haegeman A, Danchin EG, Gaur HS, Helder J, Jones MGK, Kikuchi T, Manzanilla-Lopez R, Palomares JE, Wesemael WML, and Perry RN. (2013). Top 10 plant-parasitic nematodes in molecular plant pathology. Plant Pathol: 14(9):946-61.