Zi Shi

The genome of Theobroma cacao

We sequenced and assembled the draft genome of Theobroma cacao, an economically important tropical-fruit tree crop that is the source of chocolate. This assembly corresponds to 76% of the estimated genome size and contains almost all previously described genes, with 82% of these genes anchored on the 10 T. cacao chromosomes. Analysis of this sequence information highlighted specific expansion of some gene families during evolution, for example, flavonoid-related genes. It also provides a major source of candidate genes for T. cacao improvement. Based on the inferred paleohistory of the T. cacao genome, we propose an evolutionary scenario whereby the ten T. cacao chromosomes were shaped from an ancestor through eleven chromosome fusions.

Functional analysis of the Theobroma cacao NPR1 gene in arabidopsis

BackgroundThe Arabidopsis thaliana NPR1 gene encodes a transcription coactivator (NPR1) that plays a major role in the mechanisms regulating plant defense response. After pathogen infection and in response to salicylic acid (SA) accumulation, NPR1 translocates from the cytoplasm into the nucleus where it interacts with other transcription factors resulting in increased expression of over 2000 plant defense genes contributing to a pathogen resistance response.ResultsA putative Theobroma cacao NPR1 cDNA was isolated by RT-PCR using degenerate primers based on homologous sequences from Brassica, Arabidopsis and Carica papaya. The cDNA was used to isolate a genomic clone from Theobroma cacao containing a putative TcNPR1 gene. DNA sequencing revealed the presence of a 4.5 kb coding region containing three introns and encoding a polypeptide of 591 amino acids. The predicted TcNPR1 protein shares 55% identity and 78% similarity to Arabidopsis NPR1, and contains each of the highly conserved functional domains indicative of this class of transcription factors (BTB/POZ and ankyrin repeat protein-protein interaction domains and a nuclear localization sequence (NLS)). To functionally define the TcNPR1 gene, we transferred TcNPR1 into an Arabidopsis npr1 mutant that is highly susceptible to infection by the plant pathogen Pseudomonas syringae pv. tomato DC3000. Driven by the constitutive CaMV35S promoter, the cacao TcNPR1 gene partially complemented the npr1 mutation in transgenic Arabidopsis plants, resulting in 100 fold less bacterial growth in a leaf infection assay. Upon induction with SA, TcNPR1 was shown to translocate into the nucleus of leaf and root cells in a manner identical to Arabidopsis NPR1. Cacao NPR1 was also capable of participating in SA-JA signaling crosstalk, as evidenced by the suppression of JA responsive gene expression in TcNPR1 overexpressing transgenic plants.ConclusionOur data indicate that the TcNPR1 is a functional ortholog of Arabidopsis NPR1, and is likely to play a major role in defense response in cacao. This fundamental knowledge can contribute to breeding of disease resistant cacao varieties through the application of molecular markers or the use of transgenic strategies.

Pervasive effects of a dominant foliar endophytic fungus on host genetic and phenotypic expression in a tropical tree

It is increasingly recognized that macro-organisms (corals, insects, plants, vertebrates) consist of both host tissues and multiple microbial symbionts that play essential roles in their hosts ecological and evolutionary success. Consequently, identifying benefits and costs of symbioses, as well as mechanisms underlying them are research priorities. All plants surveyed under natural conditions harbor foliar endophytic fungi (FEF) in their leaf tissues, often at high densities. Despite producing no visible effects on their hosts, experiments have nonetheless shown that FEF reduce pathogen and herbivore damage. Here, combining results from three genomic, and two physiological experiments, we demonstrate pervasive genetic and phenotypic effects of the apparently asymptomatic endophytes on their hosts. Specifically, inoculation of endophyte-free (E−) Theobroma cacao leaves with Colletotrichum tropicale (E+), the dominant FEF species in healthy T. cacao, induces consistent changes in the expression of hundreds of host genes, including many with known defensive functions. Further, E+ plants exhibited increased lignin and cellulose content, reduced maximum rates of photosynthesis (Amax), and enrichment of nitrogen-15 and carbon-13 isotopes. These phenotypic changes observed in E+ plants correspond to changes in expression of specific functional genes in related pathways. Moreover, a cacao gene (Tc00g04254) highly up-regulated by C. tropicale also confers resistance to pathogen damage in the absence of endophytes or their products in host tissues. Thus, the benefits of increased pathogen resistance in E+ plants are derived in part from up-regulation of intrinsic host defense responses, and appear to be offset by potential costs including reduced photosynthesis, altered host nitrogen metabolism, and endophyte heterotrophy of host tissues. Similar effects are likely in most plant-endophyte interactions, and should be recognized in the design and interpretation of genetic and phenotypic studies of plants.

Starch-Branching Enzyme IIa Is Required for Proper Diurnal Cycling of Starch in Leaves of Maize

Starch-branching enzyme (SBE), a glucosyl transferase, is required for the highly regular pattern of α-1,6 bonds in the amylopectin component of starch. In the absence of SBEIIa, as shown previously in the sbe2a mutant of maize (Zea mays), leaf starch has drastically reduced branching and the leaves exhibit a severe senescence-like phenotype. Detailed characterization of the maize sbe2a mutant revealed that SBEIIa is the primary active branching enzyme in the leaf and that in its absence plant growth is affected. Both seedling and mature sbe2a mutant leaves do not properly degrade starch during the night, resulting in hyperaccumulation. In mature sbe2a leaves, starch hyperaccumulation is greatest in visibly senescing regions but also observed in green tissue and is correlated to a drastic reduction in photosynthesis within the leaf. Starch granules from sbe2a leaves observed via scanning electron microscopy and transmission electron microscopy analyses are larger, irregular, and amorphous as compared with the highly regular, discoid starch granules observed in wild-type leaves. This appears to trigger premature senescence, as shown by an increased expression of genes encoding proteins known to be involved in senescence and programmed cell death processes. Together, these results indicate that SBEIIa is required for the proper diurnal cycling of transitory starch within the leaf and suggest that SBEIIa is necessary in producing an amylopectin structure amenable to degradation by starch metabolism enzymes.

Proanthocyanidin synthesis in Theobroma cacao: genes encoding anthocyanidin synthase, anthocyanidin reductase, and leucoanthocyanidin reductase.

BackgroundThe proanthocyanidins (PAs), a subgroup of flavonoids, accumulate to levels of approximately 10% total dry weight of cacao seeds. PAs have been associated with human health benefits and also play important roles in pest and disease defense throughout the plant.ResultsTo dissect the genetic basis of PA biosynthetic pathway in cacao (Theobroma cacao), we have isolated three genes encoding key PA synthesis enzymes, anthocyanidin synthase (ANS), anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR). We measured the expression levels of TcANR, TcANS and TcLAR and PA content in cacao leaves, flowers, pod exocarp and seeds. In all tissues examined, all three genes were abundantly expressed and well correlated with PA accumulation levels, suggesting their active roles in PA synthesis. Overexpression of TcANR in an Arabidopsis ban mutant complemented the PA deficient phenotype in seeds and resulted in reduced anthocyanidin levels in hypocotyls. Overexpression of TcANS in tobacco resulted in increased content of both anthocyanidins and PAs in flower petals. Overexpression of TcANS in an Arabidopsis ldox mutant complemented its PA deficient phenotype in seeds. Recombinant TcLAR protein converted leucoanthocyanidin to catechin in vitro. Transgenic tobacco overexpressing TcLAR had decreased amounts of anthocyanidins and increased PAs. Overexpressing TcLAR in Arabidopsis ldox mutant also resulted in elevated synthesis of not only catechin but also epicatechin.ConclusionOur results confirm the in vivo function of cacao ANS and ANR predicted based on sequence homology to previously characterized enzymes from other species. In addition, our results provide a clear functional analysis of a LAR gene in vivo.

The Salicylic Acid Receptor NPR3 Is a Negative Regulator of the Transcriptional Defense Response during Early Flower Development in Arabidopsis

Arabidopsis non-expressor of PR1 (NPR1) is a transcription co-activator that plays a central role in regulating the transcriptional response to plant pathogens. The NPR family consists of NPR1 and five NPR1-like genes. The NPR1 paralog NPR3 has recently been shown to function as a receptor of the plant hormone salicylic acid and to mediate proteosomal degradation of NPR1. The function of NPR3 protein during early flower development was revealed through a detailed molecular-genetic analysis including promoter transcriptional fusion analysis, phenotype characterization of npr3-3 mutants/overexpressors, and whole-plant fitness analysis. The physical interaction between NPR3 and NPR1/TGA2 was explored using bimolecular fluorescence complementation analysis in onion epidermal cells. Here, we show that NPR3 expression was strongest in the petals and sepals of developing flowers and declined after flower opening. Consistently with this observation, an npr3 knockout mutant displayed enhanced resistance to Pseudomonas syringae infection of immature flowers, but not leaves. Developing npr3 flowers exhibited increased levels of basal and induced PR1 transcript accumulation. However, the npr3 mutant showed lower fitness compared to Col-0 in the absence of pathogen. Moreover, NPR3 was shown to interact with NPR1 and TGA2 in vivo. Our data suggest that NPR3 is a negative regulator of defense responses during early flower development and it may function through the association with both NPR1 and TGA2.

TcNPR3 from Theobroma cacao functions as a repressor of the pathogen defense response

BackgroundArabidopsis thaliana (Arabidopsis) NON-EXPRESSOR OF PR1 (NPR1) is a transcription coactivator that plays a central role in regulating the transcriptional response to plant pathogens. Developing flowers of homozygous npr3 mutants are dramatically more resistant to infection by the pathogenic bacterium Pseudomonas syringae, suggesting a role of NPR3 as a repressor of NPR1-mediated defense response with a novel role in flower development.ResultsWe report here the characterization of a putative NPR3 gene from the tropical tree species Theobroma cacao (TcNPR3). Like in Arabidopsis, TcNPR3 was constitutively expressed across a wide range of tissue types and developmental stages but with some differences in relative levels compared to Arabidopsis. To test the function of TcNPR3, we performed transgenic complementation analysis by introducing a constitutively expressing putative TcNPR3 transgene into an Arabidopsis npr3 mutant. TcNPR3 expressing Arabidopsis plants were partially restored to the WT pathogen phenotype (immature flowers susceptible to bacterial infection). To test TcNPR3 function directly in cacao tissues, a synthetic microRNA targeting TcNPR3 mRNA was transiently expressed in cacao leaves using an Agrobacterium-infiltration method. TcNPR3 knock down leaf tissues were dramatically more resistance to infection with Phytophthora capsici in a leaf bioassay, showing smaller lesion sizes and reduced pathogen replication.ConclusionsWe conclude that TcNPR3 functions similar to the Arabidopsis NPR3 gene in the regulation of the cacao defense response. Since TcNPR3 did not show a perfect complementation of the Arabidopsis NPR3 mutation, the possibility remains that other functions of TcNPR3 remain to be found. This novel knowledge can contribute to the breeding of resistant cacao varieties against pathogens through molecular markers based approaches or biotechnological strategies.

Two Theobroma cacao genotypes with contrasting pathogen tolerance show aberrant transcriptional and ROS responses after salicylic acid treatment

Highlight Genotype specific strategies for plant defence: the Sca6 genotype of Theobroma cacao favors the accumulation of reactive oxygen species, while the ICS1 variety has a stronger PR gene transcriptional response.