Plant-Derived, Nodule-Specific Cysteine-Rich Peptides as a Novel Source of Biopesticides for Controlling Citrus Greening Disease.

Nodule-specific cysteine-rich (NCR) peptides, encoded in the genome of the Mediterranean legume Medicago truncatula (barrelclover), are known to regulate plant-microbe interactions. A subset of computationally derived 20-mer peptide fragments from 182 NCR peptides was synthesized to identify those with activity against the unculturable vascular pathogen associated with citrus greening disease, 'Candidatus Liberibacter asiaticus' (CLas). Grounded in a design of experiments framework, we evaluated the peptides in a screening pipeline involving three distinct assays: a bacterial culture assay with Liberibacter crescens, a CLas-infected excised citrus leaf assay, and an assay to evaluate effects on bacterial acquisition by the nymphal stage of hemipteran vector Diaphorina citri. A subset of the 20-mer NCR peptide fragments inhibits both CLas growth in citrus leaves and CLas acquisition by D. citri. Two peptides induced higher levels of D. citri mortality. These findings reveal 20-mer NCR peptides as a new class of plant-derived biopesticide molecules to control citrus greening disease.

A Perspective on Current Therapeutic Molecule Screening Methods Against 'Candidatus Liberibacter asiaticus', the Presumed Causative Agent of Citrus Huanglongbing.

Huanglongbing (HLB), referred to as citrus greening disease, is a bacterial disease impacting citrus production worldwide and is fatal to young trees and mature trees of certain varieties. In some areas, the disease is devastating the citrus industry. A successful solution to HLB will be measured in economics: citrus growers need treatments that improve tree health, fruit production, and most importantly, economic yield. The profitability of citrus groves is the ultimate metric that truly matters when searching for solutions to HLB. Scientific approaches used in the laboratory, greenhouse, or field trials are critical to the discovery of those solutions and to estimate the likelihood of success of a treatment aimed at commercialization. Researchers and the citrus industry use a number of proxy evaluations of potential HLB solutions; understanding the strengths and limitations of each assay, as well as how best to compare different assays, is critical for decision-making to advance therapies into field trials and commercialization. This perspective aims to help the reader compare and understand the limitations of different proxy evaluation systems based on the treatment and evaluation under consideration. The researcher must determine the suitability of one or more of these metrics to identify treatments and predict the usefulness of these treatments in having an eventual impact on citrus production and HLB mitigation. As therapies advance to field trials in the next few years, a reevaluation of these metrics will be useful to guide future research efforts on strategies to mitigate HLB and vascular bacterial pathogens in other perennial crops.

Interplay of Plasma Membrane and Vacuolar Ion Channels, Together with BAK1, Elicits Rapid Cytosolic Calcium Elevations in Arabidopsis during Aphid Feeding.

A transient rise in cytosolic calcium ion concentration is one of the main signals used by plants in perception of their environment. The role of calcium in the detection of abiotic stress is well documented; however, its role during biotic interactions remains unclear. Here, we use a fluorescent calcium biosensor (GCaMP3) in combination with the green peach aphid (Myzus persicae) as a tool to study Arabidopsis thaliana calcium dynamics in vivo and in real time during a live biotic interaction. We demonstrate rapid and highly localized plant calcium elevations around the feeding sites of M. persicae, and by monitoring aphid feeding behavior electrophysiologically, we demonstrate that these elevations correlate with aphid probing of epidermal and mesophyll cells. Furthermore, we dissect the molecular mechanisms involved, showing that interplay between the plant defense coreceptor BRASSINOSTEROID INSENSITIVE-ASSOCIATED KINASE1 (BAK1), the plasma membrane ion channels GLUTAMATE RECEPTOR-LIKE 3.3 and 3.6 (GLR3.3 and GLR3.6), and the vacuolar ion channel TWO-PORE CHANNEL1 (TPC1) mediate these calcium elevations. Consequently, we identify a link between plant perception of biotic threats by BAK1, cellular calcium entry mediated by GLRs, and intracellular calcium release by TPC1 during a biologically relevant interaction.

Temporal and spatial detection of Candidatus Liberibacter asiaticus putative effector transcripts during interaction with Huanglongbing-susceptible, -tolerant, and -resistant citrus hosts.

BACKGROUND: Citrus Huanglongbing (HLB) is a bacterial disease with high economic significance. The associated agent Candidatus Liberibacter asiaticus is a fastidious, phloem-limited, intracellular bacterium that is transmitted by an insect vector the Asian citrus psyllid (ACP). The genome of Ca. L. asiaticus contains protein secretion machinery that suggests host cell modulation capacity of this bacterium. RESULTS: A total of 28 candidate effectors, an important class of secreted proteins, were predicted from the Ca. L. asiaticus genome. Sequence specific primers were designed for reverse transcription (RT) and quantitative PCR (qPCR), and expression was validated for 20 of the effector candidates in infected citrus with multiple genetic background. Using detached leaf inoculation, the mRNA of effectors was detected from 6 h to 7 days post ACP exposure. It was observed that higher bacterial titers were associated with a larger number of effectors showing amplification across all samples. The effectors' expression were compared in citrus hosts with various levels of HLB tolerance, including susceptible Duncan grapefruit and Washington navel orange, tolerant citron and Cleopatra mandarin, and resistant Pomeroy trifoliate and Carrizo citrange. Across all genotypes relatively high expression was observed for CLIBASIA_03695, CLIBASIA_00460, CLIBASIA_00420, CLIBASIA_04580, CLIBASIA_05320, CLIBASIA_04425, CLIBASIA_00525 and CLIBASIA_05315 in either a host-specific or -nonspecific manners. The two genotypes in each HLB-response group also show effector-expression profiles that seem to be different. In a companion study, the expression of effectors was compared between leaves and roots of own-rooted citrus that had been Ca. L. asiaticus-infected for more than a year. Results indicated relatively high expression of CLIBASIA_03875, CLIBASIA_04800 and CLIBASIA_05640 in all leaf and some root tissues of citron, Duncan and Cleopatra. CONCLUSION: This temporal and spatial expression analysis of Ca. L. asiaticus effectors identified candidates possibly critical for early bacterial colonization, host tolerance suppression and long-term survival which are all worthy of further investigation.

Reduced Susceptibility to Xanthomonas citri in Transgenic Citrus Expressing the FLS2 Receptor From Nicotiana benthamiana.

Overexpression of plant pattern-recognition receptors by genetic engineering provides a novel approach to enhance plant immunity and broad-spectrum disease resistance. Citrus canker disease associated with Xanthomonas citri is one of the most important diseases damaging citrus production worldwide. In this study, we cloned the FLS2 gene from Nicotiana benthamiana cDNA and inserted it into the binary vector pBinPlus/ARS to transform Hamlin sweet orange and Carrizo citrange. Transgene presence was confirmed by polymerase chain reaction (PCR) and gene expression of NbFLS2 was compared by reverse transcription quantitative PCR. Reactive oxygen species (ROS) production in response to flg22Xcc was detected in transgenic Hamlin but not in nontransformed controls. Low or no ROS production was detected from nontransformed Hamlin seedlings challenged with flg22Xcc. Transgenic plants highly expressing NbFLS2 were selected and were evaluated for resistance to canker incited by X. citri 3213. Our results showed that the integration and expression of the NbFLS2 gene in citrus can increase canker resistance and defense-associated gene expression when challenged with X. citri. These results suggest that canker-susceptible Citrus genotypes lack strong basal defense induced by X. citri flagellin and the resistance of these genotypes can be enhanced by transgenic expression of the flagellin receptor from a resistant species.

Induction of innate immune responses by flagellin from the intracellular bacterium, 'Candidatus Liberibacter solanacearum'.

BACKGROUND: 'Candidatus Liberibacter solanacearum' (Lso) is a phloem-limited alphaproteobacterium associated with the devastating zebra chip disease of potato (Solanum tuberosum). Like other members of Liberibacter, Lso-ZC1 encodes a flagellin domain-containing protein (Fla Lso ) with a conserved 22 amino-acid peptide (flg22 Lso ). To understand the innate immune responses triggered by this unculturable intracellular bacterium, we studied the pathogen-associated molecular patterns (PAMPs) that triggered immunity in Nicotiana benthamiana, using the flg22 Lso peptide and the full length fla Lso gene. RESULTS: Our results showed that the expression of fla Lso via Agrobacterium-mediated transient expression induced a slow necrotic cell death in the inoculated leaves of N. benthamiana, which was coupled with a burst of reactive oxygen species (ROS) production. Moreover, the expression of several representative genes involved in innate immunity was transiently up-regulated by the flg22 Lso in N. benthamiana. The Fla Lso , however, induced stronger up-regulation of these representative genes compared to the flg22 Lso , especially that of flagellin receptor FLAGELLIN SENSING2 (FLS2) and respiratory burst oxidase (RbohB) in N. benthamiana. Although neither cell death nor ROS were induced by the synthetic flg22 Lso , a weak callose deposition was observed in infiltrated leaves of tobacco, tomato, and potato plants. CONCLUSION: The flagellin of Lso and its functional domain, flg22 Lso share characteristics of pathogen-associated molecular patterns, and trigger unique innate immune responses in N. benthamiana. Slow and weak activation of the innate immune response in host plants by the flagellin of Lso may reflect the nature of its intracellular life cycle. Our findings provide new insights into the role of the Lso flagellin in the development of potato zebra chip disease and potential application in breeding for resistance.

Aphid protein effectors promote aphid colonization in a plant species-specific manner.

Microbial pathogens and pests produce effectors to modulate host processes. Aphids are phloem-feeding insects, which introduce effectors via saliva into plant cells. However, it is not known if aphid effectors have adapted to modulate processes in specific plant species. Myzus persicae is a polyphagous insect that colonizes Arabidopsis thaliana and Nicotiana benthamiana, while the pea aphid Acyrthosiphon pisum specializes on colonizing plant species of the family Fabaceae. We found that M. persicae reproduction increased on transgenic Arabidopsis, producing the M. persicae effectors C002, PIntO1 (Mp1), and PIntO2 (Mp2), whereas reproduction of M. persicae did not increase on Arabidopsis producing the A. pisum orthologs of these three proteins. Plant-mediated RNA interference experiments showed that c002- and PIntO2-silenced M. persicae produce less progeny on Arabidopsis and N. benthamiana than nonsilenced aphids. Orthologs of c002, PIntO1, and PIntO2 were identified in multiple aphid species with dissimilar plant host ranges. We revealed high nonsynonymous versus synonymous nucleotide substitution rates within the effector orthologs, indicating that the effectors are fast evolving. Application of maximum likelihood methods identified specific sites with high probabilities of being under positive selection in PIntO1, whereas those of C002 and PIntO2 may be located in alignment gaps. In support of the latter, a M. persicae c002 mutant without the NDNQGEE repeat region, which overlaps with an alignment gap in C002, does not promote M. persicae colonization on Arabidopsis. Taken together, these results provide evidence that aphid effectors are under positive selection to promote aphid colonization on specific plant species.

A functional genomics approach identifies candidate effectors from the aphid species Myzus persicae (green peach aphid).

Aphids are amongst the most devastating sap-feeding insects of plants. Like most plant parasites, aphids require intimate associations with their host plants to gain access to nutrients. Aphid feeding induces responses such as clogging of phloem sieve elements and callose formation, which are suppressed by unknown molecules, probably proteins, in aphid saliva. Therefore, it is likely that aphids, like plant pathogens, deliver proteins (effectors) inside their hosts to modulate host cell processes, suppress plant defenses, and promote infestation. We exploited publicly available aphid salivary gland expressed sequence tags (ESTs) to apply a functional genomics approach for identification of candidate effectors from Myzus persicae (green peach aphid), based on common features of plant pathogen effectors. A total of 48 effector candidates were identified, cloned, and subjected to transient overexpression in Nicotiana benthamiana to assay for elicitation of a phenotype, suppression of the Pathogen-Associated Molecular Pattern (PAMP)-mediated oxidative burst, and effects on aphid reproductive performance. We identified one candidate effector, Mp10, which specifically induced chlorosis and local cell death in N. benthamiana and conferred avirulence to recombinant Potato virus X (PVX) expressing Mp10, PVX-Mp10, in N. tabacum, indicating that this protein may trigger plant defenses. The ubiquitin-ligase associated protein SGT1 was required for the Mp10-mediated chlorosis response in N. benthamiana. Mp10 also suppressed the oxidative burst induced by flg22, but not by chitin. Aphid fecundity assays revealed that in planta overexpression of Mp10 and Mp42 reduced aphid fecundity, whereas another effector candidate, MpC002, enhanced aphid fecundity. Thus, these results suggest that, although Mp10 suppresses flg22-triggered immunity, it triggers a defense response, resulting in an overall decrease in aphid performance in the fecundity assays. Overall, we identified aphid salivary proteins that share features with plant pathogen effectors and therefore may function as aphid effectors by perturbing host cellular processes.

Direct Infusion Device for Molecule Delivery in Plants.

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but have drawbacks, including variable uptake and the environmental breakdown of tested molecules. Trunk injection of trees is well-established, but most methods for this require expensive, proprietary equipment. To screen various treatments for Huanglongbing, a simple, low-cost method to deliver these compounds to the vascular tissue of small greenhouse-grown citrus trees infected with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) or infested with the phloem-feeding CLas insect vector Diaphorina citri Kuwayama (D. citri) is needed. To meet these screening requirements, a direct plant infusion (DPI) device was designed that connects to the plant's trunk. The device is made using a nylon-based 3D-printing system and easily obtainable auxiliary components. The compound uptake efficacy of this device was tested in citrus plants using the fluorescent marker 5,6-carboxyfluorescein-diacetate. Uniform compound distribution of the marker throughout the plants was routinely observed. Furthermore, this device was used to deliver antimicrobial and insecticidal molecules to determine their effects on CLas and D. citri respectively. The aminoglycoside antibiotic streptomycin was delivered into CLas-infected citrus plants using the device, which resulted in a reduction in the CLas titer from 2 weeks to 4 weeks post treatment. Delivering the neonicotinoid insecticide imidacloprid into D. citri-infested citrus plants resulted in a significant increase in psyllid mortality after 7 days. These results suggest that this DPI device represents a useful system for delivering molecules into plants for testing and facilitate research and screening purposes.

Impacts of Oak Mulch Amendments on Rhizosphere Microbiome of Citrus Trees Grown in Florida Flatwood Soils.

Rhizosphere interactions are an understudied component of citrus production. This is even more important in Florida flatwood soils, which pose significant challenges in achieving sustainable and effective fruit production due to low natural fertility and organic matter. Citrus growers apply soil amendments, including oak mulch, to ameliorate their soil conditions. Thus, the aim of this research was to evaluate the effects of oak mulch on citrus nutrient uptake, soil characteristics, and rhizosphere composition. The plant material consisted of 'Valencia' sweet orange (Citrus × sinensis) trees grafted on 'US-812' (C. reticulata × C. trifoliata) rootstock. The experiment consisted of two treatments, which included trees treated with oak mulch (300 kg of mulch per plot) and a control. The soil and leaf nutrient contents, soil pH, cation exchange capacity, moisture, temperature, and rhizosphere bacterial compositions were examined over the course of one year (spring and fall 2021). During the spring samplings, the citrus trees treated with oak mulch resulted in significantly greater soil Zn and Mn contents, greater soil moisture, and greater rhizosphere bacterial diversity compared to the control, while during the fall samplings, only a greater soil moisture content was observed in the treated trees. The soil Zn and Mn content detected during the spring samplings correlated with the significant increases in the diversity of the rhizosphere bacterial community composition. Similarly, the reduced rates of leaching and evaporation (at the soil surface) of oak mulch applied to Florida sandy soils likely played a large role in the significant increase in moisture and nutrient retention.

Evaluation of Spore Acquisition, Spore Production, and Host Survival Time for Tea Shot-Hole Borer, Euwallacea perbrevis, Adults after Exposure to Four Commercial Products Containing Beauveria bassiana.

Euwallacea perbrevis, the tea shot-hole borer (TSHB), is an invasive ambrosia beetle that vectors several fungal pathogens that cause Fusarium branch dieback in avocado trees in southern Florida. This study assessed the potential of four commercial products containing the entomopathogenic fungus Beauveria bassiana (Bb) for managing adult TSHB beetles. Formulated products containing Bb strains to which adult beetles were exposed were BioCeres WP, BotaniGard WP, BotaniGard ES, and Velifer ES. Controls consisted of water only and BotaniGard ES and Velifer ES supernatant with spores removed. Acquisition of spores by adult beetles dipped in product suspensions with 2.5 ± 0.1 × 106 spores/mL was assessed. Survival time of beetles after residual exposure to the Bb-based products in an in vivo avocado bark plug bioassay was determined. Production of Bb spores on beetles after being dipped in product suspensions and placed in a moistened bark-plug assay with water only was assessed. Significantly more spores were acquired by beetles exposed to Velifer ES and BotaniGard ES than beetles exposed to the other fungal products. Beetles exposed to Velifer ES and BotaniGard ES died faster (6-8 days) compared to beetles dipped in the other fungal products (10-11 days) and controls (12 days). Percentage of mycosis was highest with beetles exposed to Velifer ES (63%). Spore production on cadavers of beetles dipped in Velifer ES (20 ± 6.4 × 105 spores/cadaver) was the highest among all treatments, whereas it was the lowest on cadavers of beetles dipped in BotaniGard ES (1 ± 0.2 × 105 spores/cadaver). All Bb-based products, especially Velifer ES, demonstrated potential to manage TSHB populations under laboratory conditions. These Bb-based fungal products should be tested under field conditions to confirm these laboratory results.

Plant production of high affinity nanobodies that block SARS-CoV-2 spike protein binding with its receptor, human angiotensin converting enzyme.

Nanobodies® (VHH antibodies), are small peptides that represent the antigen binding domain, VHH of unique single domain antibodies (heavy chain only antibodies, HcAb) derived from camelids. Here, we demonstrate production of VHH nanobodies against the SARS-CoV-2 spike proteins in the solanaceous plant Nicotiana benthamiana through transient expression and their subsequent detection verified through western blot. We demonstrate that these nanobodies competitively inhibit binding between the SARS-CoV-2 spike protein receptor binding domain and its human receptor protein, angiotensin converting enzyme 2. There has been significant interest and a number of publications on the use of plants as biofactories and even some reports of producing nanobodies in plants. Our data demonstrate that functional nanobodies blocking a process necessary to initiate SARS-CoV-2 infection into mammalian cells can be produced in plants. This opens the alternative of using plants in a scheme to rapidly respond to therapeutic needs for emerging pathogens in human medicine and agriculture.

Lessons learned about the biology and genomics of Diaphorina citri infection with "Candidatus Liberibacter asiaticus" by integrating new and archived organ-specific transcriptome data.

BACKGROUND: Huanglongbing, a devastating disease of citrus, is caused by the obligate, intracellular bacterium "Candidatus Liberibacter asiaticus" (CLas). CLas is transmitted by Diaphorina citri, the Asian citrus psyllid. Development of transmission-blocking strategies to manage huanglongbing relies on knowledge of CLas and D. citri interactions at the molecular level. Prior transcriptome analyses of D. citri point to changes in psyllid biology due to CLas infection but have been hampered by incomplete versions of the D. citri genome, proper host plant controls, and/or a lack of a uniform data analysis approach. In this work, we present lessons learned from a quantitative transcriptome analysis of excised heads, salivary glands, midguts, and bacteriomes from CLas-positive and CLas-negative D. citri using the chromosomal length D. citri genome assembly. RESULTS: Each organ had a unique transcriptome profile and response to CLas infection. Though most psyllids were infected with the bacterium, CLas-derived transcripts were not detected in all organs. By analyzing the midgut dataset using both the Diaci_v1.1 and v3.0 D. citri genomes, we showed that improved genome assembly led to significant and quantifiable differences in RNA-sequencing data interpretation. CONCLUSIONS: Our results support the hypothesis that future transcriptome studies on circulative, vector-borne pathogens should be conducted at the tissue-specific level using complete, chromosomal-length genome assemblies for the most accurate understanding of pathogen-induced changes in vector gene expression.

Quercus leaf extracts display curative effects against Candidatus Liberibacter asiaticus that restore leaf physiological parameters in HLB-affected citrus trees.

Citrus greening, also called Huanglongbing (HLB), is one of the most destructive citrus diseases worldwide. It is caused by the fastidious gram-negative α-proteobacteria bacterium Candidatus Liberibacter asiaticus (CLas) and vectored by the Asian citrus psyllid (ACP), Diaphorina citri. Currently, there is no cure for HLB, no compounds have been successful in controlling HLB, and no sustainable management practices have been established for the disease. Thus, searching for alternative citrus greening disease mitigation strategies is considered an urgent priority for a sustainable citrus industry. The aim of this study was to use compounds extracted from oak, Quercus hemisphaerica, and to assess the antibacterial effects of these against CLas-infected citrus plants. The application of aqueous oak leaf extracts showed substantial inhibitory effects against CLas in citrus plants and the activity of genes related to starch. Significant differences were also observed in plant phenotypic and physiological traits after treatments. Citrus plants treated with oak extracts displayed an increase in stomatal conductance, chlorophyll content and nutrient uptake concurrently with a reduction of CLas titer, when compared to citrus plants treated with just water. The information provided from this study suggests a new management treatment program to effectively deal with the HLB disease.

LasΔ5315 Effector Induces Extreme Starch Accumulation and Chlorosis as Ca. Liberibacter asiaticus Infection in Nicotiana benthamiana.

Huanglongbing (HLB), a destructive plant bacterial disease, severely impedes worldwide citrus production. HLB is associated with a phloem-limited α-proteobacterium, Candidatus Liberibacter asiaticus (Las). Las infection causes yellow shoots and blotchy mottle on leaves and is associated with excessive starch accumulation. However, the mechanisms underlying the starch accumulation remain unknown. We previously showed that the Las5315mp effector induced callose deposition and cell death in Nicotiana benthamiana. In this study, we demonstrated that Las can experimentally infect N. benthamiana via dodder transmission. Furthermore, we revealed another key function of the Las5315 effector by demonstrating that transient expression of the truncated form of the effector, LasΔ5315, induced excessive starch accumulation by 6 fold after 8 dpi in N. benthamiana after removal of the chloroplast transit peptide from the Las5315mp. The induction mechanisms of LasΔ5315 in N. benthamiana were attributed to the up-regulation of ADP-glucose pyrophosphorylase, granule-bound starch synthase, soluble starch synthase, and starch branching enzyme for increasing starch production, and to the significant down-regulation of the starch degradation enzymes: alpha-glucosidase, alpha-amylase, and glycosyl hydrolase for decreasing starch degradation. This is the first report that Las can infect the model plant N. benthamiana. Using this model plant, we demonstrated that the LasΔ5315 effector caused the most prominent HLB symptoms, starch accumulation and chlorosis as Las infection in N. benthamiana. Altogether the Las 5315 effector is critical for Las pathogenesis, and therefore, an important target for interference.

Molecular mechanisms behind the accumulation of ATP and H2O2 in citrus plants in response to 'Candidatus Liberibacter asiaticus' infection.

Candidatus Liberibacter asiaticus (Las) is a fastidious, phloem-restricted pathogen with a significantly reduced genome, and attacks all citrus species with no immune cultivars documented to date. Like other plant bacterial pathogens, Las deploys effector proteins into the organelles of plant cells, such as mitochondria and chloroplasts to manipulate host immunity and physiology. These organelles are responsible for the synthesis of adenosine triphosphate (ATP) and have a critical role in plant immune signaling during hydrogen peroxide (H2O2) production. In this study, we investigated H2O2 and ATP accumulation in relation to citrus huanglongbing (HLB) in addition to revealing the expression profiles of genes critical for the production and detoxification of H2O2 and ATP synthesis. We also found that as ATP and H2O2 concentrations increased in the leaf, so did the severity of the HLB symptoms, a trend that remained consistent among the four different citrus varieties tested. Furthermore, the upregulation of ATP synthase, a key enzyme for energy conversion, may contribute to the accumulation of ATP in infected tissues, whereas downregulation of the H2O2 detoxification system may cause oxidative damage to plant macromolecules and cell structures. This may explain the cause of some of the HLB symptoms such as chlorosis or leaf discoloration. The findings in this study highlight important molecular and physiological mechanisms involved in the host plants' response to Las infection and provide new targets for interrupting the disease cycle.

Transient Expression of Candidatus Liberibacter Asiaticus Effector Induces Cell Death in Nicotiana benthamiana.

Candidatus Liberibacter asiaticus "Las" is a phloem-limited bacterial plant pathogen, and the most prevalent species of Liberibacter associated with citrus huanglongbing (HLB), a devastating disease of citrus worldwide. Although, the complete sequence of the Las genome provides the basis for studying functional genomics of Las and molecular mechanisms of Las-plant interactions, the functional characterization of Las effectors remains a slow process since remains to be cultured. Like other plant pathogens, Las may deliver effector proteins into host cells and modulate a variety of host cellular functions for their infection progression. In this study, we identified 16 putative Las effectors via bioinformatics, and transiently expressed them in Nicotiana benthamiana. Diverse subcellular localization with different shapes and aggregation patterns of the effector candidates were revealed by UV- microscopy after transient expression in leaf tissue. Intriguingly, one of the 16 candidates, Las5315mp (mature protein), was localized in the chloroplast and induced cell death at 3 days post inoculation (dpi) in N. benthamiana. Moreover, Las5315mp induced strong callose deposition in plant cells. This study provides new insights into the localizations and potential roles of these Las effectors in planta.

Rapid screening for citrus canker resistance employing pathogen-associated molecular pattern-triggered immunity responses.

Citrus canker, caused by the bacterial pathogen Xanthomonas citri ssp. citri (Xcc), has been attributed to millions of dollars in loss or damage to commercial citrus crops in subtropical production areas of the world. Since identification of resistant plants is one of the most effective methods of disease management, the ability to screen for resistant seedlings plays a key role in the production of a long-term solution to canker. Here, an inverse correlation between reactive oxygen species (ROS) production by the plant and the ability of Xcc to grow and form lesions on infected plants is reported. Based on this information, a novel screening method that can rapidly identify citrus seedlings that are less susceptible to early infection by Xcc was devised by measuring ROS accumulation triggered by a 22-amino acid sequence of the conserved N-terminal part of flagellin (flg22) from X. citri ssp. citri (Xcc-flg22). In addition to limiting disease symptoms, ROS production was also correlated with the expression of basal defense-related genes such as the pattern recognition receptors LRR8 and FLS2, the leucine-rich repeat receptor-like protein RLP12, and the defense-related gene PR1, indicating an important role for pathogen-associated molecular pattern-triggered immunity (PTI) in determining resistance to citrus canker. Moreover, the differential expression patterns observed amongst the citrus seedlings demonstrated the existence of genetic variations in the PTI response among citrus species/varieties.

Silencing of aphid genes by feeding on stable transgenic Arabidopsis thaliana.

Aphids are economically important pests that predominantly feed from the plant phloem. Genome, transcriptome, and proteome data are being generated for these insects, and predicted secreted proteins in aphid saliva have been identified. These secreted proteins are candidate effectors that may modulate plant processes and aid aphid colonization of plants. The next step is to develop post-genomics strategies to study the functions of identified aphid genes. One such strategy is to express aphid effector genes in planta to assess whether aphid effectors alter plant development and aphid survival and fecundity. A second strategy is to knock down the expression of aphid target genes by RNA interference (RNAi). In this chapter, we describe how to knock down aphid gene expression using plant-mediated RNAi. This strategy is useful for assessing the contribution of aphid effectors to aphid colonization of plants.

The phloem-sap feeding mealybug (Ferrisia virgata) carries 'Candidatus Liberibacter asiaticus' populations that do not cause disease in host plants.

'Candidatus Liberibacter asiaticus' (Las) is the primary causal agent of huanglongbing (HLB), the most devastating disease of citrus worldwide. There are three known insect vectors of the HLB-associated bacteria, and all are members of the Hemiptera: Diaphorina citri (Psyllidae), Trioza erytreae (Triozidae), and Cacopsylla (Psylla) citrisuga (Psyllidae). In this study, we found that another hemipteran, the striped mealybug Ferrisia virgata (Cockerell) (Hemiptera: Pseudococcidae), was able to acquire and retain Las bacteria. The bacterial titers were positively correlated with the feeding acquisition time on Las-infected leaf discs, with a two-weeks feeding period resulting in Ct values ranging from 23.1 to 36.1 (8.24 × 10(7) to 1.07 × 10(4) Las cells per mealybug). We further discovered that the prophage/phage populations of Las in the mealybugs were different from those of Las in psyllids based on Las prophage-specific molecular markers: infected psyllids harbored the Las populations with prophage/phage FP1 and FP2, while infected mealybugs carried the Las populations with the iFP3 being the dominant prophage/phage. As in the psyllids, Las bacteria were shown to move through the insect gut wall to the salivary glands after being ingested by the mealybug based on a time-course quantitative polymerase chain reaction (qPCR) assay of the dissected digestive systems. However, Las populations transmitted by the mealybugs did not cause disease in host plants. This is the first evidence of genetic difference among Las populations harbored by different insect vectors and difference among Las populations with respect to whether or not they cause disease in host plants.