LIBRO-CONGRESO-CITRUS

S09
with the aim of better understanding the infection process on citrus, we used RNA-Seq transcriptomic analysis
of infected oranges at 0, 24, 48 and 72 hours post-inoculation (hpi). Massive parallel pyrosequening with 454
FLX Titanium technology was used for sequencing the libraries. To identify the putative origin of the reads, two
reference genomes were used: (i) the Citrus sinensis Genome Assembly v1.0 (http://www.citrusgenomedb.
org/), and (ii) the P. digitatum genome, elaborated in house (submitted). Quantitative reverse transcription
PCR profiling of selected fruit and fungal genes revealed dynamic expression patterns during infection of
oranges by P. digitatum. To further investigate the putative involvement of the phenylpropanoid pathway in
the defense of citrus fruit, changes in the metabolic profile of the flavedo (outer colored part of the peel)
and albedo (inner white part) infected with P. digitatum was studied by means of HPLC-PDA-FD. Metabolite
accumulation levels along the time course suggest that flavanones, flavones, polymethoxylated flavones and
scoparone are induced in citrus fruit in response to P. digitatum infection, although with different trends
depending on the tissue.
S09P16
Disruption of the chitin synthase gene PdigChsVII in the citrus postharvest pathogen Penicillium
digitatum
Gandia M., Harries E., and Marcos J.F.
instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Food Science, Spain. mgandia@iata.csic.es
One of the main citrus postharvest pathogens is Penicillium digitatum, a citrus specific necrotrophic fungus
that penetrates fruit through injured peel and is responsible for important losses. The development of new
control methods as alternative to the current use of fungicides is urgently needed. Fungal cell wall (CW) is
composed of chitin, glucans, mannans and glycoproteins, and is considered an excellent potential target for
novel antifungals. In filamentous fungi, chitin is synthesized by a complex family of chitin synthase genes (Chs)
grouped into seven classes. We have characterized P. digitatum Chs gene expression during different axenic
growth conditions and fruit infection. Previous studies suggest a critical involvement in pathogenesis for Chs
genes of Class V and Class VII. To determine functional relevance, we have obtained deletion mutants of
PdigChsVII using Agrobacterium tumefaciens mediated transformation (ATMT). The resulting deletion strains
(ΔPdigChsVII) showed reduced growth in axenic culture, higher sensitivity to CW-interfering compounds such
as Calcofluor White (CFW) or SDS, and alterations of hyphal morphology. It also showed increase sensitivity
to reactive oxygen species (ROS) such as H2O2. Infection assays of citrus fruits with ΔPdigChsVII showed
virulence similar to the parental strain but defects in mycelium development and, importantly, also in conidia
production.
S09P17
Proteins contributing to the pathogenicity of Penicillium digitatum towards citrus fruit
Yamashita Y.K. 1 , Arimoto Y.A. 1 , Makino M.H. 2 , Annaka A.H. 1 , and Iida I.A. 1
1 Riken (Riken), Arimoto Lab., Japan; and 3 Agriculture Optical Laboratory (AOL), Plant Disease, Japan. arimoto@riken.jp
Penicillium digitatum, the causal agent of Citrus Green Mold, infects citrus fruit through injuries in the epicarp
causing damage and decay. P. digitatum is widely distributed in citrus growing areas around the world and
it is considered one of the most destructive pathogens of the citrus industry, resulting in losses of more
than half of the harvested fruits. In environments in which pH is around 5.5, P. digitatum is saprophytic and
does not invade living cells even if there is a wound. However, the fungus is pathogenic at pH lower than 3.5
and infects not only citrus fruit, but all plant cells regardless of the species. In an effort to identify proteins
contributing to pathogenicity towards citrus fruit, we performed a 2D-DIGE analysis of P. digitatum cultured
in citrus epicarp exudate adjusted to pH 5.5 and pH 3.5. At pH 3.5, we identified SOD1, AHA1, CYPA, CYPB,
and CFL as highly expressed proteins and TPIA as a low abundant protein. To investigate the contribution of
each protein to citrus fruit infectivity, we generated a series of mutants in which the gene coding for each of
these proteins was disrupted. The rate of infection of mature fruit was the same as the wild type for all the
mutants but the rate of infection in immature fruit was dramatically reduced in the Δsod1 mutant. At the side
of epicarp wounding, generation of O2 was 1.5 to 9 times greater in immature than in mature lemon fruit.
144 - VALENCIA CONFERENCE CENTER, 18th-23rd NOVEMBER 2012