Gene expression in Citrus sinensis (L.) Osbeck following infection with the bacterial pathogen Candidatus Liberibacter asiaticus causing Huanglongbing in Florida
Introduction
In August 2005 Huanglongbing (HLB), also known as “citrus greening” or “yellow shoot disease”, was discovered in Florida, USA [1], following the first report of the disease on the American continent in São Paulo State, Brazil, in 2004 [2]. HLB is a devastating disease of citrus causing the decline of trees within few years after infection and generating substantial economic losses to the citrus industry. Typical symptoms of the disease are mottling and chlorosis of the leaves along with vein-hardening, which lead to the typical appearance of yellow shoots in the tree canopy. Fruit produced on infected trees remain small, do not color properly, have a bitter taste, and often contain aborted seeds. The similarity of leaf symptoms with zinc- and other nutritional deficiencies renders the detection of HLB very difficult and requires additional analysis with molecular methods.
HLB was first reported in China in 1919, but most likely originated in Taiwan in the 1870s [3]. It is now distributed worldwide, ranging from Asia to the Indian subcontinent and neighboring islands, to Saudi Arabia, Africa, and America. The disease, which until 1970 was believed to be caused by a mycoplasma-like or viral agent, is caused by a fastidious, phloem-limited Gram-negative bacterium [4] which has not been obtained in culture as of present. Three forms of HLB are currently known. The most widespread Asian form, Candidatus Liberibacter asiaticus, is found in all HLB-affected countries except Africa. The African form, Ca. L. africanus, and the American form, Ca. L. americanus, are geographically more restricted and so far have only been found in Africa and Brazil, respectively [2], [5]. Both, Ca. L. asiaticus and Ca. L. americanus, are transmitted through Diaphorina citri Kuwayama, the Asian citrus psyllid, present in Florida since 1998, while Ca. L. africanus is transmitted through the African citrus psyllid, Trioza erytrea Del Guercio. Besides transmission through insect vectors, liberibacters can be transmitted through grafting with diseased budwood. The ability to experimentally infect periwinkle to high titer levels allowed fast progress in characterizing the bacterium phylogenetically [6] and taxonomically [7] and in developing molecular detection tools [8]. However, not much is known about the physiological or molecular processes implicated in the interaction of the bacterium with its plant host.
HLB affects all known citrus species and citrus relatives with little known resistance. Sweet oranges, mandarins and tangelos were found to be highly susceptible, followed by sour orange, grapefruits and other commercially important citrus varieties [9], [10]. Only a few lemon cultivars and a few other species like Citrus indica and Citrus macroptera reportedly displayed some tolerance or possibly resistance to the bacterium [9], [10], [11]. No known cure for HLB exists and chemical control to reduce the psyllid populations along with removal of infected trees to eliminate new sources of bacterial inoculum and the production of pathogen-free nursery plants is the current management strategy.
In response to microbial attack plants activate a series of defense responses leading to hypersensitive reaction, production of reactive oxygen species, cell wall modifications, synthesis of pathogenesis-related proteins, and accumulation of phytoalexins [12]. Disease symptom development is the result of molecular, cellular and physiological changes in a susceptible host and is part of a defense response which limits but does not restrict the spread of the pathogen in the plant [13]. Genomic techniques, such as DNA microarrays can produce valuable information on the molecular basis of plant defense responses to pathogens through the simultaneous analysis of expression patterns of thousands of genes [14]. Microarray technology has been used in numerous studies of bacterial plant diseases such as bacterial blight [15] and bacterial spot [16], as well as viral, fungal, and other diseases [17], [18]. Our objective in this study was to investigate the gene expression of sweet orange plants (Citrus sinensis L. Osbeck) in response to infection with Ca. L. asiaticus in comparison with non-infected healthy plants using the Affymetrix GeneChip® citrus genome microarray.
Due to the compatibility of the interaction significant transcriptional changes were expected to occur among genes associated with cellular changes caused by the infection process or involved in general defense and stress–responses. Disease symptom development in HLB-affected plants is associated with starch accumulation in the leaf tissue, a phenomenon which is sometimes used as a diagnostic tool [19], though it is also observed in response to nutritional deficiencies and viral infection [20], [21]. Changes in transcript levels were therefore expected to occur within the group of genes associated with carbohydrate metabolism, specifically starch synthesis.
Analyzing host plant responses at the transcriptional level is a first step in understanding the pathogenic process of HLB and may contribute to the development of new control strategies to manage this destructive disease of citrus.
Section snippets
Experiment 1
Eight greenhouse-grown 3-year-old ‘Valencia’ orange (C. sinensis) scions on Cleopatra mandarin (Citrus reticulata Blanco) rootstocks were used in the experiment. Plants were inoculated by grafting three bark pieces onto the rootstock portion of each plant. For infected plants, bark pieces were derived from ‘Lisbon’ lemon (C. limon) trees infected with Candidatus Liberibacter asiaticus from a commercial grove near Fort Pierce, St. Lucie County, Florida. Bark pieces were confirmed positive for Ca
Experiment 1
In two plants selected for microarray analysis Ca. L. asiaticus was detected 9 weeks after inoculation and remained detectable until the end of the experiment. At that time leaves began to turn yellow and displayed the characteristic mottle. Symptom development progressed quickly, and 13–17 weeks after inoculation most leaves were severely mottled. In the third plant selected for the microarray the bacterium was detected 13 and 17 weeks after inoculation at which time leaves appeared mottled.
Discussion
Citrus greening or HLB, caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus, is a devastating disease of citrus threatening all citrus-producing areas world-wide. Although the disease has been known since the 18th century, little is known about the molecular and physiological implications on its host plant. This study is a first attempt to investigate host responses and to reveal some of the mechanisms underlying disease development by monitoring transcriptional changes on
Acknowledgements
We thank G. McCollum, J. Mozoruk and B. Shatters for suggestions and critical review of the manuscript.
This research was supported in part by the Florida Citrus Production Research Advisory Council, Project No. 025-02I. Mention of a trademark, warranty, proprietary product, or vendor does not imply an approval to the exclusion of other products or vendors that also may be suitable.
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