Molecular Characterization of Disease Resistance Loci to Phytophthora palmivora in Carica papaya using AFLP Marker Analysis
In Hawaii, papaya losses due to the fungal pathogen Phytophthora palmivora can be devastating, especially during the rainy season. Of the Hawaiian papaya cultivars, 'Kamiya' is more tolerant to P. palmivora than the susceptible SunUp cultivar (Figure 1.). In an effort to understand disease tolerance and to improve papaya resistance to P. palmivora, amplified fragment length polymorphism (AFLP) molecular markers were identified between SunUp and Kamiya. The two cultivars were crossed, seeds from hermaphrodite F1 plants were sown, and the F2 progeny was grown and scored for P. palmivora disease response using a leaf disk assay. It was found that the P. palmivora tolerance in the F2 had a normal distribution, indicative of quantitative trait loci (QTL). Sixty of the most tolerant and most susceptible F2 plants were selected and analyzed using the developed AFLP markers. The markers showed that there may be polymorphisms linked with disease tolerance and disease susceptibility. Further research will be to sequence and identify the location of those markers to determine candidate genes involved in C. papaya tolerance and susceptibility to P. palmivora. These sequence characterized amplified regions (SCARs) will then be used to screen a backcross population of papayas for P. palmivora QTLs.
Mapping of QTLs is the first step toward identifying genes that are involved in P. palmivora tolerance. This approach of studying genetic regions that confer resistance to P. palmivora would make it possible breed and/or use biotechnological approaches to grow papaya varieties that are more tolerant to the fungus. This in turn will reduce crop loss and fungicide costs to farmers as well as the stress those chemicals have on Hawaii's ecosystem. Additionally, knowledge of the molecular biology will contribute to our overall objective of understanding the plant-pathogen interactions between C. papaya and P. palmivora.
Figure 1. Leaf disks of SunUp (A), Kamiya (B) and a tolerant F2 (C) 3 days after P. palmivora inoculation.
Isolating Phytophthora palmivora resistance genes from Vasconcellea goudotiana and Carica papaya 'Kamiya'
Our objective is to isolate naturally occurring resistance genes from Carica papaya 'Kamiya' and its wild relative, Vasconcellea goudotiana, that are effective in protecting papaya against the pathogenic fungus Phytophthora palmivora. These genes will be used to transform the susceptible cultivar 'SunUp', providing a new, resistant variety for producers. P. palmivora zoospore drench inoculation experiments suggest that Vasconcellea goudotiana and C. papaya 'Kamiya' possess resistance to P. palmivora that is not found in Carica papaya 'SunUp' (unpublished data, Fig. 1).
Figure 1. Root drench experiments of 'SunUp', 'Kamiya', Vasconcellea parviflora, and Vasconcellea goudotiana 3 and 7 days post-inoculation, respectively, with 1 x 103 Phytophthora palmivora zoospores/ml. The Carica papaya genome survey sequence is providing a valuable resource for genome analysis and comparison. Because Phytophthora resistance genes have already been cloned from potato and soybean, homology-based sequence comparison was initiated to search for highly related genes in papaya that may afford resistance to P. palmivora.
To do this, Solanum bulbocastanum R-gene Rpi-blb1 was compared to the Carica papaya 'SunUp' genome database. Rpi-blb1 was selected for comparison because the resistance phenotype described in potato, like the resistance seen in 'Kamiya' and V. goudotiana, does not involve a hypersensitive response (HR). Resistance afforded by Rpi-blb1 is thought to be due to limiting the rate of infection, disrupting the natural progression of disease. Interestingly, plants with this form of resistance may even allow for some pathogen sporulation and lesion development which may account for the durability of Rpi-blb1. If the pathogen is allowed to grow at some level there is likely less selection pressure to drive pathogen evolution. When V. goudotiana is inoculated with P. palmivora, some wilting and leaf loss occurs, perhaps because V. goudotiana harbors an Rpi-blb1-like homolog which limits disease progression but does not kill the pathogen (Fig. 2).
In addition, Rpi-blb1, in potato, provides broad spectrum resistance, possibly because it recognizes essential effectors common to multiple races of the pathogen. If such a gene(s) exist in 'Kamiya' or V. goudotiana, it would provide a valuable source of resistance.
Comparison of Rpi-blb1 with the Carica papaya 'SunUp' genome database revealed highest similarity to a gene from Supercontig 10, designated 10.212. Other genes from the same and other supercontigs also showed high sequence homology (Fig. 2).
Figure 2. Blastp comparison of Rpi-blb1 to predicted R-gene homologs from the Carica papaya 'SunUp' genome database and Rps1-k-1 and Rps1-k-1 from soybean. Domains were predicted using Pfam (http://pfam.sanger.ac.uk/). Red bars depict the location of leucine-rich-repeat domains (LRR). Because Supercontig 10 contains a complex R-gene homolog locus containing not only the homolog most similar to Rpi-blb1 but also the most homologs which are most similar to cloned Phytophthora R-genes, primers were designed to evaluate sequence and expression from these predicted genes. Expression comparison three days post-inoculation revealed putative induction of gene(s) from Supercontig 10 not seen using primers for similar genes located on Supercontig 16 (Fig. 4). It is possible that constitutively expressed genes from the complex locus located on Suerpcontig 16 masked induction. Nevertheless, the putative induction of an R-gene homolog from Supercontig 10 is especially noteworthy considering that the majority of Phytophthora genes cloned to date are most similar to genes from this locus. Indeed, if further investigation of this gene(s) confirms induction, immediate transformation of Carica papaya 'SunUp' should be conducted to evaluate complementation.
Cloning and sequence analysis of the putative induced gene from of V. goudotiana revealed a single gene that is related to but diverged from its counterpart in Carica papaya 'SunUp'. Multiple deletions and nucleic acid differences between V. goudotiana and Carica papaya 'SunUp' predicted gene 10.215 suggest a unique gene that, if induced, could possibly afford resistance to P. palmivora.
In addition to the identification of an induced gene from V. goudotiana, R-gene splice variants were identified from 'Kamiya' and 'SunUp'. Although R-gene splice variants have been previously identified, the abundance of splice variants in Carica papaya may explain the fewer number of R-gene homologs in Papaya relative to other plant species such as Arabidopsis and rice. Perhaps generous use of splice variants and other post-transcriptional modifications, such as RNA editing, may explain how Carica papaya is able to "get away with" only a relative hand-full of R-genes. Splice variants allow for more than one transcript from a single gene. Why splice variants are not seen for genes from Supercontig 16 is unknown. Perhaps they are less abundant, non-existent, or the primers used failed to amplify these transcripts. In summary, the presence of abundant splice variants is an exciting finding that deserves further investigation.
For more information, contact: Dr. Maureen Fitch Phone/fax: (808) 621-1375/621-1399
The aim of this project was to improve pest resistance of a commercial papaya cultivar, 'Kapoho'. A biolistic gene gun method was used to introduce the GNA gene from snowdrop (Galanthus nivalis) into papaya callus tissue. Plants were regenerated in the presence of Geneticin Disulfate salt (G418) to select for the Kanamycin selectable marker gene. Molecular analysis was performed on multiple lines to test for incorporation and expression of the transgene. Six lines were selected and further analyzed.
The level of recombinant GNA protein in each line was determined directly using an enzyme-linked immunosorbent assay (ELISA). Statistical analysis using the Statistix 7.0 program, showed that GNA expression in four of the lines was significantly different to the control Kapoho (p < 0.05). These four lines with the highest level of GNA expression were used in a laboratory bioassay using carmine spider mites, Tetranychus cinnabarinus maintained on leaf disks. It was found that mites feeding on disks from GNA-expressing plants showed a significantly lower reproductive ability (p < 0.05). Damage to leaf disks, as measured by amount of chlorophyll left in the leaf, was significantly lower compared to Kapoho (p < 0.05). Together this data suggests that expression of GNA in the leaves is affording some protection against mite feeding.
A. Leaf disk with two adult mites and eggs. B. Sample ELISA plate showing yellow colour of positive reaction.
For more information, contact: Dr. Chifumi Nagai Phone/fax: (808) 621-1385/621-1399
Musa sp. (aka Bana)
Partial Automation of Banana Micropropagation
Hawaii is the only significant commercial producer of banana (Musa sp.) in the US. Banana production in Hawaii totaled 9,250 tons in 2009. However, the Hawaiian banana industry suffers serious losses from Banana bunchy top virus (BBTV). Production of micropropagules is an important method for production of disease-free planting material. Uniformly sized micropropagated plants also have an advantage for new plantings. During the past ten years, HARC produced over 250,000 banana micropropagules using stationary liquid micropropagation protocol for all three commercial types of banana including Williams, Apple, and Saba cooking banana. Cost of production, however, is high and many small farmers cannot afford to plant micropropagules. We developed a more efficient protocol of banana micropropagation using partial automation in a large culture container system, Rita® (VITROPIC, Saint-Mathieu-Treviers, France). This method uses filtered air to push liquid medium every 4 - 6 hours into an upper chamber where propagules are placed. The HARC protocol was established by modifying the protocols used in Costa Rica and France. In vitro culture of banana was established using HARC’s stationary liquid culture system (Annual Report 1997 p.25). When the propagules started multiplying (2-3 months from culture initiation), they were placed in the Rita containers for partial immersion. We successfully established shoot multiplication in Rita® for three varieties: Saba, Apple, and William. Comparison of propagation efficiency was conducted for all three varieties. Total fresh weight and propagule numbers produced in various culture media with BA 0-10 mg/L clearly showed that the Rita system resulted more propagules within six weeks. PM2 and PM4 media produced the most efficient propagule production. The Rita system produced 30-40% higher number of shoots than those in the stationary liquid Magenta boxes. Twice as much root mass per plant was obtained in root induction medium from Rita-derived propagules as compared to those from the stationary liquid method. Comparisons of Rita and liquid Magenta cultures clearly showed that the Rita system produced higher numbers of propagules and greater fresh weight in both Apple and Williams varieties. We planted propagules of both Apple and Williams in the nursery of a commercial operation. No difference was found in survival rate (range: Apple 89%-100%, Williams 100%) or plant growth between the stationary liquid Magenta box and the Rita methods for either variety. No variant mutants were observed among the propagules multiplied in various culture media in the Rita system. The Rita method successfully lowered production cost estimates, since most of the cost of propagule production is the labor. Cost of production by the Rita system was about 30-40% less than the earlier method. Although careful handling of medium changes is required for Rita, the overall labor requirement is significantly reduced. Rita culture system however has its limitation in size. We tested 20-60 propagules per Rita container (200 ml capacity) as starting propagules. When higher numbers of propagules were placed in the container, lower multiplication and more contamination was observed. However, once scaling up of the proven Rita banana culture system is achieved, higher multiplication and overall more efficient propagule production is expected with further cost reduction.
For more information, contact: Dr. Chifumi Nagai Phone/fax: (808) 621-1385/621-1399
Anthuriums
Micropropagation
Commercial production of anthuriums would benefit from an ample supply of planting materials for new and re-planted greenhouses and shadehouses. A project was initiated to determine whether the rate of micropropagation and the number of plants that could be produced within a certain time interval could be increased. Our existing tissue culture stocks of control plants of Marian Seefurth were used as starting material. About 240 plates of Marian Seefurth were inventoried and served as stocks from which small shoot tip cuttings were used to produce new plants. Within six months, we produced about 5000 shoots from the plant clusters. Four to five months after shoot tip cuttings were prepared, about 900 of the shoots were ready to pot in the greenhouse. Losses as a result of contamination were approximately 20%, but we were able to decrease contamination by replenishing them more frequently. In early April 2008, we shipped the first samples to commercial growers to determine if the plants were suitable for planting. Without increasing the original number of stock plates, at least 10,000 shoots could be generated in one year. Increasing the stock plate number should result in even larger yields of shoots. In 2010, we shipped 4300 plants of a second cultivar provided as a micropropagated culture in 2009. In mid-2011 we shipped 3500 plants, 2800 from new callus cultures that we had initiated in 2008 to 2009. Thus, micropropagation can be carried out either with pre-existing plant cultures or by initiating callus cultures and regenerating plants. Callus and shoot cultures from about 20 different cultivars were developed and all except one were micropropagated. The exception is a cultivar obtained in 2009 that until today has not yet generated calli but we are still trying. Different growth media formulations are being evaluated to determine whether rooted shoots can be developed in less than four to five months.
Transformation for Increased Disease Resistance
Anthurium andraeanum Hort. cultivars for the cut flower market are highly susceptible to bacterial blight caused by Xanthomonas axonopodis pv. dieffenbachiae. They are also susceptible to damage from the burrowing nematode, Radopholus similis, and the root-knot nematode, Meloidogyne javanica. About 700+ independently transformed lines of two anthurium cultivars, Marian Seefurth and Midori, were obtained by our group. About 400 of the transgenic lines were shipped under USDA permit to Hilo where the tissue cultured plants were acclimatized to greenhouse conditions for screening for resistance to the pests. X. a. pv dieffenbachiae testing commenced in late March 2008 starting with 15 Marian Seefurth clones containing genes for an Arabidopsis nonpathogenesis-related protein (NPR1). NPR1 controls the cascade of gene activation events in response to pathogen attack. Clones also contained the gene attacin, a lytic peptide from the giant luna moth Hyalophora cecropia and an attacin + T4 lysozyme gene. T4 lysozyme is a lytic protein from the T4 phage virus. Three control Marian Seefurth clones were also tested. Some of the lines were tolerant to bacterial blight in green house experiments. Nematode resistance tests will be carried out on potted plants and in cinder beds infested with nematodes.
A sample of 22 transgenic lines with the NPR1 gene and six transgenic lines with the attacin gene all showed positive PCR results using primers specific for the nptII selection gene. These results show that the selection protocol is stringent. Four lines tested were all PCR positive for the attacin gene . A single band was found in lines NPR1-29-1, NPR1-31-1, NPR1-49-3, and NPR1-49-3B, indicating a single-copy transgene insertion of NPR1 into the anthurium genome . This was the first clear Southern blot result for transgenic anthurium lines produced in our lab. High quality genomic DNA extracts are critical for good PCR and Southern blot results.
Of the 700+ lines we obtained from transformation of anthurium, about three quarters contained a gene for bacterial resistance while about one quarter contained one or two nematode resistance genes (rice cystatin and/or cowpea trypsin inhibitor genes).
A different study undertaken in 2008 sought nematode resistance using RNA interference (RNAi) in which genes important to nematode health and fertility are transformed into a plant. When the pest ingests the plant substances, the transgene products stimulate nematode genes to destroy the pest's health and fertility RNAs, resulting in decreased growth, fertility, and/or death. Constructs were created by a team at Kansas State University and shipped to HARC. We have obtained about 20 transgenic lines. PCR determinations for selection and transgenes in the first putative line are to be completed shortly.
Transformation for Novel Flower Color
Growth in the floriculture industry is enhanced by the creation of new shapes, colors, and fragrances in traditional blossoms. The introduction of a blue rose by Suntory company of Japan inspired many to dream of other blue flowers. Why not anthurium? We are collaborating with a researcher in Britain and with University of Hawaii researchers to create such an anthurium. We wish to produce a purple or blue, large, heart-shaped anthurium for the local industry. A set of genes in the anthocyanin biosynthetic pathway will be used. We will observe transient expression of the genes by bombarding them into anthurium spathes of different colors and will also stably transform cultivars expressing a range of spathe colors. The time span from inserting the genes into anthurium to first bloom is approximately 28 months.
For more information, contact: Dr. Maureen Fitch Phone/fax: (808) 621-1375/621-1399
For information on, or the possibility of buying excess stocks of plants, please contact Maureen Fitch ([email protected]).