Parasitic plant genes necessary

for haustorium initiation

John Yoder

University of California-Davis

So many genes,

so little time

John Yoder

University of California-Davis

How to make sense of all these genes

Triphysaria

Young, Steiner, dePamphilis 1999

Triphysaria is a

facultative

hemiparasite in the

Orobanchaceae

Heide-Jorgesen and Kuijt (1995) The

haustorium of the root parasite

Triphysaria with special reference the

xylem bridge ultrastructure. Am J Bot

“The haustorium

is the organ which

embodies the

very idea of

parasitism”

(Kuijt 1969)

Triphysaria forms lateral haustoria on a wide

range of host plants

ArabidopsisMaize

Grass Lupine

Haustoria are induced by contact with host

roots, host root exudates and purified

haustorium initiation factors

peonidin DMBQ

Genes co-regulate by Arabidopsis roots and DMBQ

P-Glycoprotein

Glytatione-S-transferase

Ribosomal Protein (loading

control)

30 uM DMBQArabidopsis roots

0h 0.5h 2h 5h 24h 0h 0.5h 2h 5h 24h

EDIT13H_Pl_4

Translation initiation factor

Late embriogenesis

abundant protein

Transcriptionally

controlled tumor protein

Wound induced

cytochrome P450

D HP E EtT

EDIT Host Forward

D H PEEt T

1)Vacuum infiltration

Root

regeneration

Gene transformation of

Triphysaria roots

2)Agrobacterium tumefaciens

callus transformation

3) Agrobacterium rhizogenes

root transformation

•Method first developed for

nodulation studies Medicago

(Boisson-Dernier, Barker 2001)

•MSU440 strain

•Acetosyringone in media

•Fluorescence selection, not antibioitics

2 weeks old seedlings 10 days after transformation

Agrobacteium rhizogenes transformation of

Triphysaria roots

0

Days after transformation

10 30 205

2 week old seedlings

Day 0

3 months

YFP selection

Cut off roots

Dip cut site in Agrobacterium

Array in media plates

Transgenic Triphysaria roots are capable of

forming haustoria

fluorescencebright field

yellow fluorescence protein (YFP)beta-glucuronidase (GUS)

bright field fluorescence

PDK Intronhp hp t-OCSCaMV 35SYFPMAS

Functional characterization; RNAi silencing using

Gateway technology

pHG8-YFP

Target for silencing

machinery

Transcription

PDK IntronccdB ccdBt-OCSCaMV 35SYFPMAS

pHpGW-Cloning

ggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggg329 aaHuman

CRYZ

Catalyze one electron reductions

TvQR1 encodes a single electron

reducing QR homologous to human

zeta crystallin

+ 1 e

Plant Journal 2001

PcQR 201 aa

Catalyzes two electron reduction of 1,4-benzoquinone

gggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggg

TvQR2 encodes a two electron reducing QR with

homology in Phanerochaete chrysosporium

+ 2 e

Plant Phys 2001

TvQR1 and TvQR2 transcripts are rapidly up-

regulated by allelopathic quinones

AtQR1 is

downregulated in

Arabidopsis

0 2 X 0 2 X 0 2n 2r X

T.v. T.e. T.p.

Plant Journal 2001

TvQR1 t-OCSCaMV 35S TvQR1

TvQR2 t-OCSCaMV 35S TvQR2

TvQR2+TvQR1CaMV 35S TvQR1+TvQR2 t-OCS

Hairpin constructions transformed into Triphysaria

roots reduced transcript levels of TvQR1 and TvQR2

TvQR1 TvQR2Plant Cell 2010

Silencing of TvQR1 but not TvQR2

reduces haustorium development

Plant Cell 2010

Pirin is a

transcription

factor binding

protein

1

10

100

1000

Water treated DMBQ treated Host treated

Re

lati

ve

ex

pre

ssio

n l

ev

els

A

TvPIRIN is up-regulated by DMBQ and

host root contact

1

10

100

1000

pHG8-YFP pHpPRN

Relative transcripts levels

TvPIRIN

C

Water treatment DMBQ treatment

RNAi lowers TvPIRIN transcript levels and

haustorium development

Transcripts Haustorium development

Current model of haustorium

initiation

Transcription

factors

Pirinauxin

Contributors

Yoder lab

Pradeepa Gunathilake (PhD)

Alexey Tomilov (post doc)

Natalya Tomilova (post doc)

Tatiana Fillapova (technician)

Denneal Jamison (PhD)

Manuel Torres (PhD)

Marta Matvienko (post doc))

Biao Wu (post doc)

Jim Westwood (VTI)

Mike Timko (UV)

Claude DePamphillis (PSU)

Norm Wickett (PSU)

Loren Honaas (PSU)

Richard Michelmore (UCD)

Tadeusz Wroblewski (UCD)

John Bowman (Monash)

Ericka Lindquist (JGI)

Corina Antonescu (TIGR)

Collaborators