Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
Citrus Research and Education Center
Non Technical Summary
Citrus greening is considered the most serious disease in citrus worldwide, and has now reached a state of epidemic in Florida, resulting in the loss of $9 billion and over 8,000 jobs. This disease has recently been detected in California, and control strategies are crucial to prevent the collapse of the citrus industry in the US and worldwide. The disease is caused by several members of bacterial pathogens in the genus Liberibacter, that are efficiently transmitted from infected to healthy plants by psyllids, a group of plant sap-sucking insects. The control of these diseases relies on using chemical insecticides for controlling the insect vector populations, but these are still ineffective, and thus the development of sustainable control measures is urgently needed. Here I will study the factors that play a role in the bacteria movement inside the citrus plants and inside the insect vector. I will identify plant and insect genes involved in the plant-pathogen and insect-pathogen interactions, study their role in the bacterial movement, and manipulate their function for investigating their potential as targets for preventing the transmission and spread of the pathogens. The outcomes of this research will enable the development of an efficient control mechanism for this group of pathogens.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
0%
Goals / Objectives
Citrus greening has now reached a state of epidemic in Florida, resulting in the loss of $7.8 billion and over 7,000 jobs, and putting the future of Florida's citrus industry at risk. This devastating disease has now been detected in California, and control strategies are crucial to prevent the collapse of the citrus industry in the US. It is caused by the phloem-restricted bacteria Candidatus Liberibacter asiaticus (CLas), and is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri. A promising approach to prevent the pathogen transmission is to interfere with the the plant-pathogen and vector-pathogen interactions, but our understanding of these processes is very limited. Moreover, we still do not understand how the bacteria move and spread inside the plant and the psyllid.In the plant CLas localize and propagate inside the phloem cells, and therefore will move between cells through the symplamic connection (i.e. sieve pores and plasmodesmata). However, there is very little knowledge about CLas movement. Association of callose with sieve areas and sieve plates in angiosperms has been widely demonstrated . It was shown that the accumulation of callose in the wall sheath around the channel results in decreased cell-to-cell movement of florescent dyes , while treatments inhibiting callose deposition resulted in an increased diameter of plasmodesmata (Pd) orifices and a higher Pd size exclusion limit (SEL) . Similar results were shown in sieve elements. Induced phloem callose led to a decrease in the lateral movement of C14-assimilates and auxin, while treatments that stimulate breakdown of sieve plate callose led to increased movement of fluorescein through the sieve tubes . Recently, a phloem-specific Callose synthase (CalS7) was identified, and its absence resulted in carbohydrate starvation . These results suggest that the presence of basal levels of callose is actually required for efficient carbohydrate transport in the phloem, and point to a more complex relationship between sieve pore callose and phloem transport, where both too much and too little callose will have a negative effect. Remarkably, it was shown that callose accumulation during CLas infection impaired symplastic dye movement into the vascular tissue, and inhibited photoassimilate export in the infected leaves , strongly suggesting that similar mechanisms take place during CLas infection.CLas is transmitted by psyllids in a persistent manner. CLas was detected in various ACP organs, including the salivary glands, hemolymph, filter chamber, midgut, fat and muscle tissues, and ovaries , suggesting that CLas propagates within the insect tissues. Previous research has suggested that CLas multiplies in nymphal stages of the ACP but not in adults , and in order to be efficiently transmitted by adults it has to be acquired in the nymphal stage and to replicate and reach sufficient amounts for transmission. Transmission rates of the CLas and CLso probably depend on the ability of the bacteria to multiply within insect tissue and reach sufficient titers for transmission, and on the ability to cross barriers during the transmission pathway especially the gut-hemolymph and the hemolyph-salivary glands barriers. The mechanism of this movement is still unknown.The goal of this work is to expand our knowledge regarding the spread of CLas inside the plant and its insect vector, and to establish novel strategies to decrease the disease symptoms of HLB infected plant by removing the callose plugs and restoring sugar transport. The specific objectives are:(a) Identify Pd associated beta-1,3-glucanases in citrus, and utilizing it to regulate callose levels.(b) Measure the sieve elements callose levels in synaptotagmins and remorins silenced plants or plants treated with auxin.(c) Study the effect of Synaptotagmins on CLas exit from ACP gut cells.
Project Methods
(a) Identification of Pd associated beta-1,3-glucanases in citrus. In Citrus sinesis, at least 33 transcripts encoding for glucan endo-1,3-β-glucosidase exist (https://phytozome.jgi.doe.gov/pz/portal.html). Using the BIG-PI Plant Predictor (http://mendel.imp.ac.at/gpi/plant_server.html), a tool that was developed to specifically identify GPI modification site prediction in plants, I have identified 12 transcripts encoding for β-1,3-glucanase proteins that are GPI anchored, two of the contain only the GH17 domain (orange1.1g043682m, orange1.1g015263m), and the other ten contain both the GH17 and X8 domains (orange1.1g010142m, orange1.1g011131m, orange1.1g011636m orange1.1g011391m, orange1.1g010782m, orange1.1g010931m, orange1.1g011532m, orange1.1g048257m , orange1.1g045344m, orange1.1g010789m). As a first step, my goal is to identify which of these 12 candidates encodes for a Pd related enzymes. Each sequence will be synthesized in such a way that the green fluorescent protein (GFP) will be located just before the GPI omega cleavage site, as described in (Levy et al., 2007; Moghadam and Jackson, 2013). These constructs will then be precipitated into the surface of the gold particles and transiently expressed in Citrus sinesis leaves by bombardment. Leaves will be analyzed by confocal scanning microscopy to identify fusion proteins that localize as puctae pattern in the cell periphery, the characteristic Pd pattern. Pd localization will be verified by staining leaf sections with aniline blue, to label Pd callose, as described in (Levy et al., 2007). Those β-1,3-glucanases that will be identified to be Pd-localized will then be cloned, without the GFP, into the PacI-StuI sited in CTV for future analysis. This is a very straightforward approach that have already been successful in tobacco (Bucher et al., 2001).(b) Measure the sieve elements callose levels in synaptotagmins and remorins silenced plants or plants treated with auxin. Citrus sinesis encodes for six synaptotagmins and eleven remorins. In collaboration with Dr. Gowda from the CREC, we have already constructed CTV-based silencing constructs for these genes. The synaptotagmin CTV clone contains nucleotides 563-941from orange1.1g009307m, which shares 90% homology to Arabidopsis SYTA. The remorin silencing CTV clone contains nucleotides 1257-1531 of orange1.1g009448m, which represents the area with the highest homology to the other members of the remoin family. These CTV clones have been already generated, semi-purified from N. benthamiana infected plants, and are now ready for citrus inoculation. Citrus macrophylla will be inoculated with these constructs, as described in (Hajeri et al., 2014). I will use quantitative RT-PCR in order to identify silenced plants. Callose levels in silenced/unsilenced leaves sieve elements will be determined by aniline blue staining, either as a result of stress (wound) or without stress. In order to avoid stress-induced callose leaves will be fixed immediately by either freezing with dry-ice or immersing in 98% ethanol. Callose levels will be determined as described in (Levy et al., 2007). In addition, I will also compare stress-induced callose by applying IAA (auxin) solution into a wound in the leaf, and compare callose levels to plants wounded without IAA.(c) Effect of Synaptotagmins on CLas exit from ACP gut cells. A bioinformatics analysis identified three clear Synaptotagmin soding sequences in the ACP genome-synaptotagmin 1-like (LOC103512870), synaptotagmin-7 (LOC103520142) and synaptotagmin-like protein 5 (LOC103516817). We will employ the CTV-based RNAi system, developed by Drs. Bill Dawson and Siddarame Gowda. I will generate CTV-RNAi constructs to silence Synaptotagmin genes in ACP. A sequence of 300 to 400 nucleotides from the corresponding gene targets RNAs. The generated CTV clone will be first agro-infiltrated into Nicotiana benthamiana plants, to establish infection. CTV virions will be isolated from systemic infected leaves of N. benthamiana, and will be used to infect Citrus macrophylla. Silencing ACP will be performed by rearing ACP nymphs on the C. macrophylla plants that were infected with the various VIGS inducing viruses, as described in (Hajeri et al., 2014). Adult psyllids will be collected and their silenced gene expression will be determined by qRT-PCR. As controls, ACPs will be reared on C. macrophylla plants infected with CTV carrying an empty vector. The accumulation of CLas in the silenced psyllids will be determined using both FISH in dissected guts, and qPCR of gut DNA. I will look for changes in bacteria accumulation levels between treated and control treated (untreated) psyllids. In addition, the longevity, fecundity, and fertility of the treated psyllids will be determined as well. Lastly, I will compare the ability of these psyllids to transmit the bacteria to healthy plants. Treated or control treated adult psyllids that carry the Liberibacter will be placed on healthy plants. After 15 days all adults and nymphs will be removed so that only eggs will be left, and new emerging adults will be collected. The transmission of the bacteria into these new adults will be determined by both quantitative PCR and FISH.Ammar, E.-D., Shatters, R.G., Jr., and Hall, D.G. (2011). Localization of Candidatus Liberibacter asiaticus, Associated with Citrus Huanglongbing Disease, in its Psyllid Vector using Fluorescence in situ Hybridization. Journal of Phytopathology 159, 726-734.Bucher, G.L., Tarina, C., Heinlein, M., Di Serio, F., Meins, F., Jr., and Iglesias, V.A. (2001). Local expression of enzymatically active class I beta-1, 3-glucanase enhances symptoms of TMV infection in tobacco. Plant J 28, 361-369.Hajeri, S., Killiny, N., El-Mohtar, C., Dawson, W.O., and Gowda, S. (2014). Citrus tristeza virus-based RNAi in citrus plants induces gene silencing in Diaphorina citri, a phloem-sap sucking insect vector of citrus greening disease (Huanglongbing). Journal of Biotechnology 176, 42-49.Levy, A., Erlanger, M., Rosenthal, M., and Epel, B.L. (2007). A plasmodesmata-associated beta-1,3-glucanase in Arabidopsis. Plant J 49, 669-682.Moghadam, P.K., and Jackson, M.B. (2013). The Functional Significance of Synaptotagmin Diversity in Neuroendocrine Secretion. Frontiers in Endocrinology 4, 124.Pelz-Stelinski, K.S., Brlansky, R.H., Ebert, T.A., and Rogers, M.E. (2010). Transmission Parameters for Candidatus Liberibacter asiaticus by Asian Citrus Psyllid (Hemiptera: Psyllidae). Journal of Economic Entomology 103, 1531-1541.