Dynamics of 'Candidatus Liberibacter asiaticus' Growth, Concentrations of Reactive Oxygen Species, and Ion Leakage in Huanglongbing-Positive Sweet Orange.

Citrus Huanglongbing (HLB) caused by 'Candidatus Liberibacter asiaticus' (CLas) is the most devastating citrus disease worldwide. CLas induces systemic and chronic reactive oxygen species (ROS) production, which has been suggested to be a primary cause of cell death in phloem tissues and subsequent HLB symptoms. Mitigating oxidative stress caused by CLas using horticultural approaches has been suggested as a useful strategy to reduce HLB damages. To provide information regarding the application timing to mitigate ROS, we investigated monthly dynamics of CLas concentration, CLas-triggered ROS, and phloem cell death in the bark tissues of asymptomatic and symptomatic branches of HLB-positive Hamlin and Valencia sweet orange trees in the field. Healthy branches in the screenhouse were used as controls. CLas concentration exhibited significant variations over the course of the year, with two distinct peaks observed in Florida citrus groves-late spring/early summer and late fall. Within both Hamlin and Valencia asymptomatic tissues, CLas concentration demonstrated a negative correlation with the deviation between the monthly average mean temperature and the optimal temperature for CLas colonization in plants (25.7°C). However, such a correlation was not evident in symptomatic tissues of Hamlin or Valencia sweet oranges. ROS levels were consistently higher in symptomatic or asymptomatic branches than in healthy branches in most months. ROS concentrations were higher in symptomatic branches than in asymptomatic branches in most months. CLas triggered significant increases in ion leakage in most months for asymptomatic and symptomatic branches compared with healthy controls. In asymptomatic branches of Hamlin, a positive correlation was observed between CLas concentration and ROS concentrations, CLas concentration and ion leakage levels, as well as ROS and ion leakage. Intriguingly, such a relationship was not observed in Valencia asymptomatic branches or in the symptomatic branches of Hamlin and Valencia. This study sheds light on the pathogenicity of CLas by providing useful information on the temporal dynamics of ROS production, phloem cell death, and CLas growth, as well as provides useful information in determining the timing for application of antioxidants and antimicrobial agents to control HLB.

Zinkicide Is a ZnO-Based Nanoformulation with Bactericidal Activity against Liberibacter crescens in Batch Cultures and in Microfluidic Chambers Simulating Plant Vascular Systems.

Phloem-limited bacterial "Candidatus Liberibacter" species are associated with incurable plant diseases worldwide. Antimicrobial treatments for these pathogens are challenging due to the difficulty of reaching the vascular tissue they occupy at bactericidal concentrations. Here, in vitro antimicrobial mechanisms of Zinkicide TMN110 (ZnK), a nonphytotoxic zinc oxide (ZnO)-based nanoformulation, were compared to those of bulk ZnO (b-ZnO) using as a model the only culturable species of the genus, Liberibacter crescens Minimum bactericidal concentration (MBC) determination and time-kill assays showed that ZnK has a bactericidal effect against L. crescens, whereas b-ZnO is bacteriostatic. When ZnK was used at the MBC (150 ppm), its antimicrobial mechanisms included an increase in Zn solubility, generation of intracellular reactive oxygen species, lipid peroxidation, and cell membrane disruption; all of these were of greater intensity than those of b-ZnO. Inhibition of biofilms, which are important during insect vector colonization, was stronger by ZnK than by b-ZnO at concentrations between 2.5 and 10 ppm in batch cultures; however, neither ZnK nor b-ZnO removed L. crescens preformed biofilms when applied between 100 and 400 ppm. In microfluidic chambers simulating source-to-sink phloem movement, ZnK significantly outperformed b-ZnO in Zn mobilization and bactericidal activity against L. crescens planktonic cells in sink reservoirs. In microfluidic chamber assays assessing antibiofilm activity, ZnK displayed a significantly enhanced bactericidal activity against L. crescens individual attached cells as well as preformed biofilms compared to that of b-ZnO. The superior mobility and antimicrobial activity of ZnK in microenvironments make this formulation a promising product to control plant diseases caused by "Candidatus Liberibacter" species and other plant vascular pathogens.IMPORTANCE "Candidatus Liberibacter" species are associated with incurable plant diseases that have caused billions of dollars of losses for United States and world agriculture. Chemical control of these pathogens is complicated, because their life cycle combines intracellular vascular stages in plant hosts with transmission by highly mobile insect vectors. To date, "Candidatus Liberibacter" species are mostly unculturable, except for Liberibacter crescens, a member of the genus that has been used as a model for in vitro assays. Here, we evaluated the potential of Zinkicide (ZnK) as an antimicrobial against "Candidatus Liberibacter" species in batch cultures and under flow conditions, using L. crescens as a biological model. ZnK displayed bactericidal activity against L. crescens in batch cultures and showed increased mobility and bactericidal activity in microfluidic devices resembling "Candidatus Liberibacter" species natural habitats. ZnK performance observed here against L. crescens makes this compound a promising candidate to control plant diseases caused by vascular pathogens.

Distinct microbial communities among different tissues of citrus tree Citrus reticulata cv. Chachiensis.

Plant microbiota colonize all organs of a plant and play crucial roles including supplying nutrients to plants, stimulating seed germination, promoting plant growth, and defending plants against biotic and abiotic stress. Because of the economic importance, interactions between citrus and microbes have been studied relatively extensively, especially citrus-pathogen interactions. However, the spatial distribution of microbial taxa in citrus trees remains under-studied. In this study, Citrus reticulata cv. Chachiensis was examined for the spatial distribution of microbes by sequencing 16S rRNA genes. More than 2.5 million sequences were obtained from 60 samples collected from soil, roots, leaves, and phloem. The dominant microbial phyla from all samples were Proteobacteria, Actinobacteria and Acidobacteria. The composition and structure of microbial communities in different samples were analyzed by PCoA, CAP, Anosim and MRPP methods. Variation in microbial species between samples were analyzed and the indicator microbes of each sample group were identified. Our results suggested that the microbial communities from different tissues varied significantly and the microenvironments of tree tissues could affect the composition of its microbial community.

Dynamics of Candidatus Liberibacter asiaticus Movement and Sieve-Pore Plugging in Citrus Sink Cells.

Citrus greening or Huanglongbing (HLB) is caused by the phloem-limited intracellular Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). HLB-infected citrus phloem cells undergo structural modifications that include cell wall thickening, callose and phloem protein induction, and cellular plugging. However, very little is known about the intracellular mechanisms that take place during CLas cell-to-cell movement. Here, we show that CLas movement through phloem pores of sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi) is carried out by the elongated form of the bacteria. The round form of CLas is too large to move, but can change its morphology to enable its movement. CLas cells adhere to the plasma membrane of the phloem cells specifically adjacent to the sieve pores. Remarkably, CLas was present in both mature sieve element cells and nucleated nonsieve element cells. The sieve plate plugging structures of host plants were shown to have different composition in different citrus tissues. Callose deposition was the main plugging mechanism in the HLB-infected flush, where it reduced the open space of the pores. In the roots, pores were surrounded by dark extracellular material, with very little accumulation of callose. The expression of CALLOSE SYNTHASE7 and PHLOEM PROTEIN2 genes was upregulated in the shoots, but downregulated in root tissues. In seed coats, no phloem occlusion was observed, and CLas accumulated to high levels. Our results provide insight into the cellular mechanisms of Gram-negative bacterial cell-to-cell movement in plant phloem.

Challenging battles of plants with phloem-feeding insects and prokaryotic pathogens.

For the past 4 decades, intensive molecular studies of mostly leaf mesophyll cell-infecting pathogens and chewing insects have led to compelling models of plant-pathogen and plant-insect interactions. Yet, some of the most devastating pathogens and insect pests live in or feed on the phloem, a systemic tissue belonging to the plant vascular system. Phloem tissues are difficult to study, and phloem-inhabiting pathogens are often impossible to culture, thus limiting our understanding of phloem-insect/pathogen interactions at a molecular level. In this Perspective, we highlight recent literature that reports significant advances in the understanding of phloem interactions with insects and prokaryotic pathogens and attempt to identify critical questions that need attention for future research. It is clear that study of phloem-insect/pathogen interactions represents an exciting frontier of plant science, and influx of new scientific expertise and funding is crucial to achieve faster progress in this important area of research that is integral to global food security.

Fourier-transform infrared (FT-IR) spectroscopy analysis discriminates asymptomatic and symptomatic Norway spruce trees.

Conifer trees, including Norway spruce, are threatened by fungi of the Heterobasidion annosum species complex, which severely affect timber quality and cause economic losses to forest owners. The timely detection of infected trees is complicated, as the pathogen resides within the heartwood and sapwood of infected trees. The presence of the disease and the extent of the wood decay often becomes evident only after tree felling. Fourier-transform infrared (FT-IR) spectroscopy is a potential method for non-destructive sample analysis that may be useful for identifying infected trees in this pathosystem. We performed FT-IR analysis of 18 phloem, 18 xylem, and 18 needle samples from asymptomatic and symptomatic Norway spruce trees. FT-IR spectra from 1066 - 912 cm-1 could be used to distinguish phloem, xylem, and needle tissue extracts. FT-IR spectra collected from xylem and needle extracts could also be used to discriminate between asymptomatic and symptomatic trees using spectral bands from 1657 - 994 cm-1 and 1104 - 994 cm-1, respectively. A partial least squares regression model predicted the concentration of condensed tannins, a defense-related compound, in phloem of asymptomatic and symptomatic trees. This work is the first to show that FT-IR spectroscopy can be used for the identification of Norway spruce trees naturally infected with Heterobasidion spp.

Growth parameters of Liberibacter crescens suggest ammonium and phosphate as essential molecules in the Liberibacter-plant host interface.

BACKGROUND: Liberibacter crescens is the closest cultured relative of four important uncultured crop pathogens. Candidatus. L. asiaticus, L. americanus, L. africanus cause citrus greening disease, while Ca. L. solanacearum causes potato Zebra chip disease. None of the pathogens grows in axenic culture. L. crescens grows in three media: a BM-7, a serum-free Hi® Grace's Insect Medium (Hi-GI), and a chemically-defined medium called M15. To date, no optimal growth parameters of the model species L. crescens have been reported. Studying the main growth parameters of L. crescens in axenic culture will give us insights into the lifestyle of the Ca. Liberibacter pathogens. RESULTS: The evaluation of the growth parameters-pH, aeration, temperature, and buffering capacity-reflects the optimal living conditions of L. crescens. These variables revealed that L. crescens is an aerobic, neutrophilic bacterium, that grows optimally in broth in a pH range of 5.8 to 6.8, in a fully oxygenated environment (250 rpm), at 28 °C, and with monosodium phosphate (10 mM or 11.69 mM) as the preferred buffer for growth. The increase of pH in the external media likely results from the deamination activity within the cell, with the concomitant over-production of ammonium in the external medium. CONCLUSION: L. crescens and the Ca. Liberibacter pathogens are metabolically similar and grow in similar environments-the phloem and the gut of their insect vectors. The evaluation of the growth parameters of L. crescens reveals the lifestyle of Liberibacter, elucidating ammonium and phosphate as essential molecules for colonization within the hosts. Ammonium is the main driver of pH modulation by active deamination of amino acids in the L. crescens amino acid rich media. In plants, excess ammonium induces ionic imbalances, oxidative stress, and pH disturbances across cell membranes, causing stunted root and shoot growth and chlorosis-the common symptoms of HLB-disease. Phosphate, which is also present in Ca. L. asiaticus hosts, is the preferred buffer for the growth of L. crescens. The interplay between ammonium, sucrose, potassium (K+), phosphate, nitrate (NO3-), light and other photosynthates might lead to develop better strategies for disease management.

Fungal canker pathogens trigger carbon starvation by inhibiting carbon metabolism in poplar stems.

Carbon starvation is the current leading hypothesis of plant mortality mechanisms under drought stress; recently, it is also used to explain tree die-off in plant diseases. However, the molecular biology of the carbon starvation pathway is unclear. Here, using a punch inoculation system, we conducted transcriptome and physiological assays to investigate pathogen response in poplar stems at the early stages of Botryosphaeria and Valsa canker diseases. Transcriptome assays showed that the majority of differentially expressed genes (DEGs) in stem phloem and xylem, such as genes involved in carbon metabolism and transportation, aquaporin genes (in xylem) and genes related to the biosynthesis of secondary metabolites and the phenylpropanoid pathway (related to lignin synthesis), were downregulated at 7 days after inoculation (DAI). Results also showed that the expression of the majority of disease-resistance genes upregulated in poplar stems, which may be connected with the downregulation expression of the majority of WRKY family genes. Physiological assays showed that transpiration rate decreased but WUE (water use efficiency) increased the 3 and 7 DAI, while the net photosynthetic rate decreased at 11 DAI in Botryosphaeria infected poplars (ANOVA, P < 0.05). The NSC (non-structural carbohydrates) content assays showed that the soluble sugar content of stem phloem samples increased at 3, 7, and 11 DAI that might due to the impede of pathogen infection. However, soluble sugar content of stem xylem and root samples decreased at 11 DAI; in contrast, the starch content unchanged. Therefore, results revealed a chronological order of carbon related molecular and physiological performance: declination of genes involved in carbon and starch metabolism first (at least at 7 DAI), declination of assimilation and carbon reserve (at 11 DAI) second. Results implied a potential mechanism that affects the host carbon reserve, by directly inhibiting the expression of genes involved in carbon metabolism and transport.

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.

High-Throughput Sequencing Analysis of the Bacterial Community in Stone Fruit Phloem Tissues Infected by "Candidatus Phytoplasma prunorum".

"Candidatus Phytoplasma prunorum" (CPp) is a highly destructive phytopathogenic agent in many stone fruit-growing regions in Europe and the surrounding countries. In this work, we focused on documenting entire bacterial community in the phloem tissues of 60 stone fruit trees. Nested PCR and two real-time PCR assays were used to select CPp-positive (group A) and CPp-negative samples (group B). Afterwards, high-throughput amplicon sequencing was performed to assess bacterial community compositions in phloem tissues. The bacterial composition in phloem tissue consisted of 118 distinct genera, represented mainly by Pseudomonas, Acinetobacter, Methylobacterium, Sphingomonas, and Rhizobium. Statistics showed that CPp influenced the bacterial composition of infected plants (group A) and that the bacterial community depended on the geographical origin of the sample. This is the first work focusing on an analysis of the influence of CPp on the bacteria coexisting in the phloem tissues of stone fruit trees.

Phytoplasmas: An Introduction.

Phytoplasmas are among the most recently discovered plant pathogens. They are wall-less prokaryotes restricted to phloem tissue, associated with diseases affecting several hundred plant species. The impact of phytoplasma diseases on agriculture is impressive and, at the present day, no effective curative strategy has been developed. The availability of rapid and sensitive techniques for phytoplasma detection as well as the possibility to study their relationship with the host plants is a prerequisite for the management of phytoplasma-associated diseases.

Comparison of Different Procedures for DNA Extraction for Routine Diagnosis of Phytoplasmas.

This chapter presents five different procedures for extracting DNA from phytoplasma-infected plants and insect vectors suitable for PCR assays. One of these procedures enriches phytoplasma DNA through differential centrifugation and is effective in producing highly purified DNA from fresh tissues from a wide variety of herbaceous and woody plants. Although the DNA yield is less than those of other known total DNA extraction procedures, a major advantage of the presented phytoplasma-enriched procedure is that a substantial proportion of the isolated DNA is from phytoplasmas. The other four procedures here presented involve treatments with CTAB-based buffer to lyse cells and purify DNA followed by deproteination and recovery of DNA. These procedures work well for extracting total DNA from fresh, frozen, or lyophilized tissues from a wide variety of plant hosts as well as insect vectors. Because few manipulations are required, the CTAB-based procedures are faster and easier to perform than the phytoplasma-enrichment protocol. In addition, they result in very high yields and provide DNA that is less pure but of suitable quality for the use in standard molecular biological techniques including PCR assays.

Real-Time PCR Protocol for Phytoplasma Detection and Quantification.

Phytoplasmas are mollicutes restricted to plant phloem tissue and are normally present at very low concentrations. Real-time polymerase chain reaction (qPCR) offers several advantages over conventional PCR. It is a fast, sensitive, and reliable detection technique amenable to high throughput. Two fluorescent chemistries are available, intercalating dyes or hybridization probes. Intercalating dyes are relatively less expensive than TaqMan® hybridization probes but the latter chemistry is the most commonly used for phytoplasma detection. qPCR may be designed for universal detection of phytoplasma, group or subgroup specific detection, or for simultaneous detection of up to three or four phytoplasmas (multiplexing). qPCR may be used for relative or absolute quantification in host plants and in insect vectors. Therefore, qPCR plays an important role in phytoplasma detection as well as in host-pathogen interaction and in epidemiological studies. This chapter outlines the protocols followed in qPCR assay for phytoplasma detection and quantification, focusing mainly on the use of TaqMan® probes.

Sieve Elements: The Favourite Habitat of Phytoplasmas.

The sieve elements are the only plant compartments, where phytoplasmas can survive and propagate. Therefore, this chapter is focussed on the specific molecular and cell-biological properties of the sieve element. Sieve element-companion cell complexes arise from (pro)cambial mother cells induced by key genes known to be decisive for sieve-element differentiation. The special anatomy, cell biology, and plasma-membrane outfit of sieve elements allows them to act collectively as a tube system that is able to drive a mass flow against the flow induced by transpiration. Plasmodesmal corridors are vital for the translocation of photoassimilates and systemic signals and for survival of the enucleate sieve elements. Of paramount importance is the Ca2+-dependent gating of plasmodesmata by callose and proteins. Hence, some of the complex, regulatory mechanisms to maintain Ca2+ homoeostasis in sieve elements are presented. Finally, the peculiarities of the chemical and physical sieve-element environment offered to phytoplasmas are discussed.

Laser Microdissection of Phytoplasma-Infected Grapevine Leaf Phloem Tissue for Gene Expression Study.

Phytoplasmas have been found confined mainly in leaf phloem sieve elements. In spite of this, few researches have been focused on the infected phloem tissue, whereas the plant response at the infection site could be quite different compared to distal parts and almost completely masked when whole organs are considered. Herein, we provide a protocol for the isolation of leaf phloem from paraffin-embedded samples by Laser Microdissection, followed by RNA purification and RNA amplification to generate cDNA libraries. Our protocol, which has been set up for phytoplasma-infected field-grown grapevine and successfully used for gene expression profiling, can be modified according to different plant species.

DAPI and Confocal Laser-Scanning Microscopy for In Vivo Imaging of Phytoplasmas.

As phytoplasmas are located inside the phloem tissue, always surrounded by numerous layers of other cells, they can result difficult candidates for microscopical investigations. Moreover, the necessity to kill the plant tissues for microscopy observations causes instantaneous and irreversible modifications in the sieve elements, leading to misleading information and erroneous interpretations. Phytoplasmas were here investigated in intact Vicia faba host plants using DAPI as fluorescent probe and confocal laser scanning microscopy. The described nondestructive technique may be applied for the imaging of phytoplasmas and of different pathogen-related responses in planta.

Application of laser microdissection to study phytoplasma site-specific gene expression in the model plant Arabidopsis thaliana.

Many aspects of plant diseases caused by phytoplasmas are still unknown, as these pathogens are phloem restricted, uncultivable wall-less bacteria and must be studied always in association with their host. Phytoplasma transcripts are strongly underrepresented within host tissues and this poses problems for gene expression analyses. In this study, a procedure was established to infect the model plant Arabidopsis thaliana with the phytoplasma Flavescence dorée, a serious threat to European viticulture. Rates of phytoplasma infective insects and transmission efficiency to A. thaliana as well as pathogen loads were measured in different tissues of infected A. thaliana plants, and modification of phloem cell ultrastructure was observed in infected plant tissues at microscopic level. Moreover, a protocol for the application of laser microdissection to analyze plant and phytoplasma gene expression profiles in the specific colonized tissue was designed. The procedure allowed a good preservation of the plant tissue anatomy. Results showed that the extracted RNA was suitable for qualitative and quantitative RT-PCR, since both plant and pathogen transcripts, either abundant or rare ones, could be detected without any pre-amplification step. The combined use of laser microdissection approach and A. thaliana to study phytoplasmas opens the way to exploit biological, molecular and bioinformatic tools available for the model plant and to elucidate key pathways of the infection mechanisms of these important plant pathogen.

Plant growth-promoting bacteria Kosakonia radicincitans mediate anti-herbivore defense in Arabidopsis thaliana.

MAIN CONCLUSION: This study demonstrates that the application of the PGPB strain, Kosakonia radicincitans enhances a plant's resistance against phloem-feeding and chewing insects in Arabidopsis thaliana. The plant growth-promoting bacterial strain K. radicincitans DSM 16656 applied to A. thaliana reduced the number of phloem-feeding insects of both the specialist Brevicoryne brassicae and the generalist Myzus persicae. While weight gain of the generalist chewing insect Spodoptera exigua was reduced by 30% on A. thaliana plants treated with K. radicincitans, growth of the specialist caterpillar Pieris brassicae was not affected when compared with caterpillars from control plants. Since generalist and specialist chewing insects responded differentially to PGPB application, the implication of signaling pathways in PGPB mediated changes in plant defense was studied using two signaling pathway mutants impaired in their salicylic acid (npr1-1 mutant) or jasmonic acid (coi1-1 mutant) pathway. We found that the jasmonic acid pathway is relevant for upregulation of aliphatic glucosinolates and suppression of the chewing generalist S. exigua larval growth. Chewing from generalist P. brassicae increased glucosinolate content in A. thaliana leaves mediated via both signaling pathways. However, only in the npr1-1 mutant, which contains the highest aliphatic glucosinolate content, the P. brassicae induced further enrichment of glucosinolates, resulting in a reduction of larval growth. Effects of K. radicincitans on plant resistance could not be explained by changes in glucosinolate contents or composition. Our results demonstrate the distinct role played by K. radicincitans in suppressing insect performance in A. thaliana.

Integrated Phloem Sap mRNA and Protein Expression Analysis Reveals Phytoplasma-infection Responses in Mulberry.

To gain insight into the response of mulberry to phytoplasma-infection, the expression profiles of mRNAs and proteins in mulberry phloem sap were examined. A total of 955 unigenes and 136 proteins were found to be differentially expressed between the healthy and infected phloem sap. These differentially expressed mRNAs and proteins are involved in signaling, hormone metabolism, stress responses, etc. Interestingly, we found that both the mRNA and protein levels of the major latex protein-like 329 (MuMLPL329) gene were increased in the infected phloem saps. Expression of the MuMLPL329 gene was induced by pathogen inoculation and was responsive to jasmonic acid. Ectopic expression of MuMLPL329 in Arabidopsis enhances transgenic plant resistance to Botrytis cinerea, Pseudomonas syringae pv tomato DC3000 (Pst. DC3000) and phytoplasma. Further analysis revealed that MuMLPL329 can enhance the expression of some defense genes and might be involved in altering flavonoid content resulting in increased resistance of plants to pathogen infection. Finally, the roles of the differentially expressed mRNAs and proteins and the potential molecular mechanisms of their changes were discussed. It was likely that the phytoplasma-responsive mRNAs and proteins in the phloem saps were involved in multiple pathways of mulberry responses to phytoplasma-infection, and their changes may be partially responsible for some symptoms in the phytoplasma infected plants.

MiRNA-seq-based profiles of miRNAs in mulberry phloem sap provide insight into the pathogenic mechanisms of mulberry yellow dwarf disease.

A wide range of miRNAs have been identified as phloem-mobile molecules that play important roles in coordinating plant development and physiology. Phytoplasmas are associated with hundreds of plant diseases, and the pathogenesis involved in the interactions between phytoplasmas and plants is still poorly understood. To analyse the molecular mechanisms of phytoplasma pathogenicity, the miRNAs profiles in mulberry phloem saps were examined in response to phytoplasma infection. A total of 86 conserved miRNAs and 19 novel miRNAs were identified, and 30 conserved miRNAs and 13 novel miRNAs were differentially expressed upon infection with phytoplasmas. The target genes of the differentially expressed miRNAs are involved in diverse signalling pathways showing the complex interactions between mulberry and phytoplasma. Interestingly, we found that mul-miR482a-5p was up-regulated in the infected phloem saps, and grafting experiments showed that it can be transported from scions to rootstock. Based on the results, the complexity and roles of the miRNAs in phloem sap and the potential molecular mechanisms of their changes were discussed. It is likely that the phytoplasma-responsive miRNAs in the phloem sap modulate multiple pathways and work cooperatively in response to phytoplasma infection, and their expression changes may be responsible for some symptoms in the infected plants.