A Cauliflower Mosaic Virus Effector Protein that Manipulates Multiple Defence Pathways in Arabidopsis

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Dr. Muhammad Shafiq

Assistant Professor TTS

Institute of Agricultural Sciences

University of the Punjab Lahore

• Plants must continuously defend themselves against attacks from bacteria, viruses, fungi, invertebrates, and even other plants.
• The long-term goal of crop improvement for biotic stress tolerance in plants is a traditional objective of breeders.

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• Specificity of defense mechanisms, specificity of parasitic ability, inheritance of resistance, gene-for-gene interaction, and durability of resistance.
• Major considerations in breeding for resistance to parasites, conventional sources of resistance and possible alternatives, namely mutation breeding, genetic manipulations, tissue cultures, and molecular interventions to develop plants resistant to pests and pathogens will also be dealt.

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Host vs non-Host Resistance

• Plants are continually exposed to a vast number of potential pathogens and, as a result, they have evolved intricate defense mechanisms to recognize and defend themselves against a wide array of these disease-causing agents by inducing a set of defense responses that can defeat the invading pathogens. These responses include a hypersensitive response (HR; rapid localized cell death at the site of infection), increased expression of defense-related genes [e.g. pathogenesis-related (PR) genes], and the oxidative burst.
• Often, the plant disease resistance described is cultivar or accession specific and is referred to as host resistance

• A second resistance, operating under less-understood mechanisms, provides resistance against pathogens throughout all members of a plant species. This type of resistance is referred to as nonhost resistance.
• A pathogen that cannot cause disease on a nonhost plant is referred to as a nonhost pathogen.

Key Point the talk

• Effective genetic and molecular mechanisms that plants have developed to recognize and respond to infection by a number of pathogens and pests,
• nonhost resistance,
• constitutive barriers
• race-specific resistance,
• including recent advances in elucidating the structure and molecular mechanisms used by plants to cope with pathogens and pest attacks.

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�Plant Pathologists’ Joke!�

Dozens if you are Pseudomonas syringae

Hundreds if you are an oomycete

For a virus, one will do the trick

A:

A Model Host-Virus Pathosystem

Mechanically inoculate with CaMV

3 weeks later

Cauliflower mosaic virus replicates and spreads systemically in nearly all ecotypes of Arabidopsis

Transcripts of SA-responsive marker genes (PR-1, BGL2, PR-5) increase in abundance from about 8 dpi

Love et al. 2005, Plant Physiology 139: 935-948

CaMV infection stimulates multiple defence responses in Arabidopsis

CaMV DNA

The situation is more complex than it appears!

SA-mediated defence responses are capable of restricting virus spread but may be ineffective in wild-type plants.

Expression patterns of defence-related genes show spatial and temporal complexities not apparent from whole plant studies!

• CaMV induces systemic SA-signaling responses but these are inhibited in tissues invaded by virus

• Is one of the virus proteins acting as a defence suppressor?

• ORF VI encodes a 520 αα polypeptide P6/TAV that fulfils an essential role in virus replication and is required for translation of the 35S RNA

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• ORF VI is the main genetic determinant of pathogenicity

• ORF VI is the main genetic determinant of pathogenicity for CaMV

CaMV is a pararetrovirus with a circular dsDNA genome of ~8030 bp.

The link between ORF VI and virus pathogenicity make P6 an obvious suspect as an effector protein

P6 is a multifunctional protein with several domains

1 100 200 300 400 500

NES

miniTAV

RNA-a

RNA-b

Assy

Adapted from Kobayashi and Hohn 2003 J. Virol 77:8557 & 2004 MPMI 17:475, Haas et al. 2005 Plant Cell17:927

Zn

F

Transgenic Arabidopsis lines that express P6 constitutively from a 35S promoter

Pr-35S

CaMV ORF VI

OCS-Ter

Cecchini et al. 1997, MPMI 10, 1094

P6 transgenic lines are resistant to the auxin transport inhibitor TIBA

P6 transgenic lines show little or no ethylene triple response

Love et al. 2005 Plant Physiol 139:935, Geri et al. Plant Mol Biol 56:111, Love et al. 2007 J. Gen Virol (in press)

Transgenic Arabidopsis lines that express P6 show pleiotropic alterations in signaling

P6 is a suppressor of RNA silencing

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P6 suppresses PR-1 expression in CaMV-infected Arabidopsis

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CaMV

Mock

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Does P6 block signalling up- or downstream of SA?

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SA

Gene Expression

PR-1 etc

CaMV

Treat P6-transgenic and WT plants with SA.

Do they differ in the expression of SA-responsive genes?

Host receptor

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P6 suppresses responses downstream of SA

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P6 suppresses cell death induced by SA-treatment

• Both gene-for-gene and basal defence responses are affected

If P6 suppresses SA signalling, does expression affect susceptibility to other biotrophic pathogens?

Does P6 also affect responses upstream of SA?

P6 down-regulates SA but up-regulates JA

Does P6 affect JA-signaling?

• NPR1 is an ankyrin-repeat, BTB-POZ domain protein that is required for transcriptional activation of many SA-responsive genes (e.g. PR-1). Microarray studies suggest that NPR1 is involved in regulating expression of >1000 genes in response to SA

What common components regulate responses to SA and JA?

• NPR1 modulates cross-talk between SA and JA signalling pathways

• NPR1 is a key central regulator of defence!

Could NPR1 be a target for P6?

Does P6 regulate transcription of NPR1?

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Transgenics expressing a NPR1:GFP fusion protein* can be used to monitor NPR1 localization in response to SA.

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We crossed the 35S::NPR1:GFP transgene into a 35S::P6 background and compared localization of GFP after SA treatment

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* Mou et al., 2003 Cell 113:935.

Does P6 affect NPR1 at the protein level?

Time after SA treatment (mins)

How does P6 affect NPR1?

PR gene

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Our data are consistent with a model in which P6 modulates JA- and SA-signaling through its effect on NPR1

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P6

JA biosynthesis/ signaling

Promoter

bZIP

bZIP

Conclusions

• P6 is also an efficient suppressor of RNA-silencing – could suppression of defence responses and suppression of silencing be linked?

• P6 promotes the modification of NPR1 to an inactive form that is nuclear localized, leading to changes in gene expression and defence signaling.

• SA-signaling is suppressed but JA-signaling is enhanced.

• Auxin and Ethylene signaling are affected through cross-talk with SA

• P6 acts as an effector protein, facilitating systemic spread CaMV by suppressing SA-mediated defence responses that would otherwise restrict long-distance movement.