Wednesday March 31

Parallel session 5: Fungal Way of Living: Sex and Other Encounters

PS5.1

The dodge of blotch: Saving sex in Mycosphaerella graminicola
Gerrit HJ Kema¹, SB Ware^1,2, TAJ van der Lee¹, AHJ Wittenberg^1,3, C Diaz¹, SB Goodwin⁴, MA de Waard⁵, S. Ben M’Barek^1
1Plant Research International, Wageningen UR, PO box 69, 6700AB Wageningen, the Netherlands
²Current address: Ball Horticultural Company – Worldwide Headquarters, 622 Town Road, West Chicago, Illinois 60185, USA ³Current address: Keygene N.V., Agro Business Park 90, 6708 PW Wageningen, the Netherlands
⁴USDA-ARS, 915 West State Street, Purdue University, West Lafayette, IN, USA ⁵Wageningen University, Laboratory of Phytopathology, P.O. Box 8025, 6700 EE Wageningenthe Netherlands

gert.kema@wur.nl

Mycosphaerella graminicola is the causal agent of septoria tritici blotch, currently the most important disease of wheat in Europe. Despite the recent identification of 15 resistance genes and their potential application in plant breeding, disease control is currently achieved mainly by fungicides. However, fungicide resistance development in natural M. graminicola populations frequently occurs and is a serious concern. Depending on the fungicides this may develop gradually, such as with resistance to azoles, or much more rapidly as was observed for strobilurin fungicides. In order to understand this rapid spread of resistance we have performed a range of crossing experiments that demonstrate that external stress factors hamper disease development but cannot prevent sexual development. As M. graminicola is a heterothallic bipolar pathogen, sexual development requires two mating partners - carrying different mat alleles (mat1-1 or mat 1-2) - that both produce female and male organs. We use an in planta crossing protocol that reliably enables the isolation of segregating/mapping populations. The first stress factor that we used was host resistance. Various crosses on a range of cereal hosts indicated that sex always takes place as long as one of the mating partners is virulent. Thus, even an avirulent isolate that does not establish a compatible interaction with the host plant is perfectly able to enter into the sexual process resulting in viable ascospores. As a consequence the genes of such an avirulent isolate are transmitted to subsequent generations. This is fundamentally different from many other host-pathosystems where avirulent isolates - and their genes - are lost in subsequent generations. We used strobilurin fungicides as a second stress factor by crossing sensitive and resistant isolates under various strobilurin concentrations (3-200%). Although strobilurins prevent disease development of sensitive isolates, and as a consequence minimize biomass, abundant sexual development occurred under all conditions, thus irrespective of the applied strobilurin concentration. Moreover, our results showed that the ‘stressed’ mating partner – the sensitive parent – acted as the preferred paternal partner. Thus, external stress factors on avirulent or sensitive isolates do not preclude the production of M. graminicola spermatia that effectuate viable ascospore production. The fact that the sensitive isolates are preferred paternal donors – and consequently the resistant strains are maternal donors – in the sexual process resulted in major shifts in strobilurin resistance in the segregating populations as the target site for strobilurins is on the mitochondrial genome. A minimal dose of 6% strobilurin already rendered entire populations resistant to these compounds. This explains the rapid pan-European spread of strobilurin resistance in M. graminicola, likely in temporally and geographically independent occasions, with no loss of nuclear genetic variation. The recently discovered genome plasticity of M. graminicola may contribute to its ability to overcome environmentally adverse conditions.

PS5.2

Chasing effectors in the genomes of fungal symbionts and pathogens of trees

Sébastien Duplessis

INRA, France

duplessi@nancy.inra.fr

After the completion of the Populus trichocarpa genome sequence (Tuskan et al, Science, 2006), the Joint Genome Institute (JGI, Department of Energy, USA) sequenced several genomes of microbes interacting with poplar trees. This program comprises fungi with different lifestyles (symbionts and pathogens) and aims at understanding their role in forest ecosystems and molecular mechanisms underlying tree-microbe interactions. Analysis of the coding space of genomes of the ectomycorrhizal basidiomycete Laccaria bicolor (sequenced by the JGI, Martin et al, Nature, 2008) and the ectomycorrhizal ascomycete Tuber melanosporum (the gourmet black truffle, sequenced by the french Genoscope) revealed that the two fungi have derived different ‘symbiosis molecular toolkits’ to associate with their hosts. Interestingly, L. bicolor genome encodes numerous small secreted proteins that share motifs with effectors recently described in fungal and oomycete pathogens. Analysis of the genome of Melampsora larici-populina, the basidiomycete responsible for the poplar rust disease (sequenced by JGI) also revealed the presence of a large repertoire of small secreted protein encoding genes that likely contains putative effectors required to establish successful colonization of plant tissues. Examples from these fungal genomic projects will illustrate how genomic analyses combined with transcriptomic approaches helped in identifying candidate effectors and ongoing functional characterization of candidates in both L. bicolor and M. larici-populina will be presented.

PS5.3

Investigating the genetic control of infection-related development in the rice blast fungus Magnaporthe oryzae

Nicholas J. Talbot, Diane G. O Saunders, Michael, J. Kershaw, Martin J. Egan, Romain Huguet, Ana-Lilia Martinez-Rocha, Yasin Dagdas, Min He

School of Biosciences, University of Exeter, Geoffrey Pope Building, Exeter EX4 4QD, United Kingdom. E-mail: n.j.talbot@exeter.ac.uk

Magnaporthe oryzae is the causal agent of rice blast, one of the most serious economic problems affecting rice production.  The availability of genome sequences for M. oryzae and its host, Oryza sativa, has provide the means to investigate this fungal-plant interaction in great detail and develop a system biology approach to understanding plant disease. During plant infection, M. oryzae develops a differentiated infection structure called an appressorium. This unicellular, dome-shaped structure generates cellular turgor, that is translated into mechanical force to cause rupture of the rice cuticle and entry into plant tissue. My research group is interested in determining the molecular basis of appressorium development and understanding the genetic regulation of the infection process by the rice blast fungus. We have shown that development of a functional appressorium is linked to the control of cell division. Blocking completion of mitosis by generation of a temperature-sensitive monimA mutant, for instance, prevents appressorium morphogenesis and a similar phenotype occurs when MobimE mutants are analysed. Furthermore, following mitosis, conidia undergo cell collapse and programmed autophagic cell death.  The absence of non-selective autophagic cell machinery in M. oryzae is sufficient to prevent the fungus from being able to cause disease. These findings indicate that appressorium morphogenesis requires completion of mitosis and initiation of autophagic recycling of the contents of the fungal spore to the appressorium.  Appressorium formation is also associated with an oxidative burst that requires NADPH oxidases that a virulence determinants of M. oryzae. To study appressorium physiology and function in greater detail we have used proteomics to define the major changes in protein abundance associated with plant infection by M. oryzae and metabolite fingerprinting by electrospray ionisation mass spectrometry and GC-ToF-MS to define major metabolic changes in both the fungus and its host during the onset of rice blast disease. This is linked to our study of the physiology of turgor generation and the role of glycerol, trehalose and glycogen metabolism to the production of infection-competent apppressoria.

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