# Functional analysis of cassiicolin, effector of the rubber tree pathogen Corynespora cassiicola.

Sébastien Ribeiro, Marine Deon, Dinh Minh Tran, Mouman Soumahoro, Aurélien Masson, André Clément-Demange, Valérie Pujade-Renaud

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Sébastien Ribeiro, Marine Deon, Dinh Minh Tran, Mouman Soumahoro, Aurélien Masson, et al. Functional analysis of cassiicolin, effector of the rubber tree pathogen Corynespora cassiicola. 12th EFPP-10th SFP conference: Deepen Knowledge in Plant Pathology for Innovative Agro-Ecology, May 2017, Dunkerque, France. pp.A0. hal-01605689

HAL Id: hal-01605689

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Submitted on 5 Jun 2020

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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Sébastien Ribeiro<fnref n="1" />,<fnref n="2" />, Marine Déon<fnref n="1" />,<fnref n="2" />, Dinh Minh Tran<fnref n="2" />,<fnref n="3" />, Mouman Soumahoro<fnref n="4" />, Aurélien Masson<fnref n="5" />, André Clément-Demange<fnref n="2" />, Valérie Pujade-Renaud<fnref n="1" />,<fnref n="2" />

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

## Background

Rubber tree (*Hevea brasiliensis*) is an economically important tropical tree that provides natural rubber. In Asia and Africa, it is affected by the *Corynespora Leaf Fall (CLF) disease* caused by the *necrotrophic fungus Corynespora cassiicola*. On the most susceptible rubber clones, *C. cassiicola* can cause massive defoliation leading yield losses. Some pathogenic strains secrete a small protein toxin, the *cassiicolin*<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, which is transcriptionally up-regulated during early stages of the disease<fnref n="4" />. Six isoforms were identified and used to classify the various strains into **toxin classes<fnref n="5" />**. However, *C. cassiicola* strains without cassiicolin gene (toxin class Cas0) may also produce toxic exudates⁶, suggesting the existence of effectors other than cassiicolin.

<figure><img src="image_4.png" /><figcaption>susceptible clone</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>tolerant clone</figcaption></figure>

## Objective

To determine the **importance of cassiicolin Cas1 for virulence of C. cassiicola**,

by comparing the wild-type virulent CCP strain and the same strain deleted for the *cassiicolin-encoding gene Cas1*, in interaction with rubber tree.

## Construction a *Cas1* gene deletion mutant

Deletion mutant was obtained by replacing the *Cas1* gene from a *C. cassiicola* highly aggressive strain (**CCP**) with a hygromycin resistance cassette by homologous recombination. The deletion mutant was named **ccpΔcas1**.

<figure><img src="image_6.png" /><figcaption>hygromycin cassette</figcaption></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>wild-type
CCP genome</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>deletion mutant
ccpΔcas1 genome</figcaption></figure>

## Comparison between CCP and *ccpΔcas1*

The growth rate, conidia production and percentage of germination were compared *in vitro* between **CCP** and **ccpΔcas1** strains:

<table><thead><tr><th>Strain</th><th>Growth rate (mm/day)</th><th>Conidiation (conidia/µl)</th><th>Germination (%)</th></tr></thead><tbody><tr><td>CCP</td><td>4.13 ± 0.16a</td><td>413 ± 77a</td><td>71.75 ± 9a</td></tr><tr><td>ccpΔcas1</td><td>4.29 ± 0.08a</td><td>479.5 ± 42a</td><td>69.75 ± 6a</td></tr></tbody></table>

→ Deletion of the *Cas1* gene did not modify major physiological functions in the CCP strain.

## Toxicity of *ccpΔcas1* filtrate

Eighteen rubber clones were tested for their sensitivity to *C. cassiicola* exudates, by conductivity measurement of the induced electrolyte leakage (%EL)⁶.

Hierarchical clustering =
Clones (CL) x Treatments (Tr)

Treatments:

Culture filtrates (strain name - toxin class)

Purified cassiicolin at various concentration (Tox1, 5 or 10)

Blanks (Tox0 or culture medium Cz)

<figure><img src="image_10.png" /></figure>

→ **Both CCP and *ccpΔcas1* filtrates are toxic, although with different profiles:**

CCP is clustered in Tr2, with Tox5 and Tox10

*ccpΔcas1* is clustered in Tr4, with Cas0 strains

## Pathogenicity of *ccpΔcas1* strain

CCP and *ccpΔcas1* strains were compared for their virulence on detached leaves of three rubber clones, 4 days after application of conidia (50 conidia/µl, 6 drops per leaves):

<figure><img src="image_11.png" /></figure>

→ Without *cassiicolin Cas1*, the CCP strain is almost avirulent and doesn't extend beyond the inoculation spot.

## Conclusion

*Cassiicolin Cas1 is the major virulence factor involved in the compatible interaction between CCP and rubber clones*, despite the presence of other putative effectors causing filtrate toxicity.

## REFERENCES

1. BRETON, F. (2000) Journal of Natural Rubber Research 3, 115-128.

2. BARTHE, P. (2007) Journal of Molecular Biology 367, 89-1013.

3. DE LAMOTTE, F. (2007) Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 849, 357-362.

4. DÉON, M. (2012a) Plant science: an international journal of experimental plant biology 185-186, 227-237.

5. DÉON, M. (2014) Fungal Biology 118, 32-47.

6. TRAN, D.M. (2016) PLOS ONE 11, e0162807. Public Library of Science.

<footnote n="1">¹Université Clermont Auvergne (UCA), UMR 547 PIAF, BP 10448, F-63000 Clermont-Ferrand, France</footnote>

<footnote n="2">²CIRAD, UMR AGAP, F-34398 Montpellier, France</footnote>

<footnote n="3">³Rubber Research Institute of Vietnam, Ho Chi Minh City, Vietnam</footnote>

<footnote n="4">⁴Mouman Soumahoro, Société Africaine de Plantations d’Hévéa, Côte d’Ivoire</footnote>

<footnote n="5">⁵Société des Caoutchoucs de Grand-Béréby, Côte d’Ivoire</footnote>