Comparative Metabolome and Transcriptome Analyses Reveal Differential Enrichment of Metabolites with Age in Panax notoginseng Roots.

Panax notoginseng is a perennial plant well known for its versatile medicinal properties, including hepatoprotective, antioxidant, anti-inflammatory, anti-tumor, estrogen-like, and antidepressant characteristics. It has been reported that plant age affects the quality of P. notoginseng. This study aimed to explore the differential metabolome and transcriptome of 2-year (PN2) and 3-year-old (PN3) P. notoginseng plant root samples. Principal component analysis of metabolome and transcriptome data revealed major differences between the two groups (PN2 vs. PN3). A total of 1813 metabolites and 28,587 genes were detected in this study, of which 255 metabolites and 3141 genes were found to be differential (p < 0.05) between PN2 vs. PN3, respectively. Among differential metabolites and genes, 155 metabolites and 1217 genes were up-regulated, while 100 metabolites and 1924 genes were down-regulated. The KEGG pathway analysis revealed differentially enriched metabolites belonging to class lipids ("13S-hydroperoxy-9Z, 11E-octadecadionic acid", "9S-hydroxy-10E, 12Z-octadecadionic acid", "9S-oxo-10E, 12Z-octadecadionic acid", and "9,10,13-trihydroxy-11-octadecadionic acid"), nucleotides and derivatives (guanine and cytidine), and phenolic acids (chlorogenic acid) were found to be enriched (p < 0.05) in PN3 compared to PN2. Further, these differentially enriched metabolites were found to be significantly (p < 0.05) regulated via linoleic acid metabolism, nucleotide metabolism, plant hormone signal transduction, and arachidonic acid metabolism pathways. Furthermore, the transcriptome analysis showed the up-regulation of key genes MAT, DMAS, SDH, gallate 1-beta-glucosyltransferase, and beta-D-glucosidase in various plants' secondary metabolic pathways and SAUR, GID1, PP2C, ETR, CTR1, EBF1/2, and ERF1/2 genes observed in phytohormone signal transduction pathway that is involved in plant growth and development, and protection against the various stressors. This study concluded that the roots of a 3-year-old P. notoginseng plant have better metabolome and transcriptome profiles compared to a 2-year-old plant with importantly enriched metabolites and genes in pathways related to metabolism, plant hormone signal transduction, and various biological processes. These findings provide insights into the plant's dynamic biochemical and molecular changes during its growth that have several implications regarding its therapeutic use.

Accumulation and Transmission of 'Candidatus Liberibacter solanacearum' Haplotypes by the Nymphs of Two Psyllid Vectors.

'Candidatus Liberibacter solanacearum' (Lso) is a plant pathogenic bacterium transmitted by psyllids that causes significant agricultural damage. Several Lso haplotypes have been reported. Among them, LsoA and LsoB are transmitted by the potato psyllid Bactericera cockerelli and infect solanaceous crops, and LsoD is transmitted by the carrot psyllid B. trigonica and infects apiaceous crops. Several studies evaluated the transmission of these haplotypes by adult psyllids. However, fewer data are available on the transmission of different Lso haplotypes by psyllid nymphs. In this study, we investigated the transmission of these three haplotypes by psyllid nymphs to expand our basic understanding of Lso transmission. Specifically, the objective was to determine if the haplotypes differed in their transmission rates by nymphs and if LsoA and LsoB accumulated at different rates in the guts of nymphs as it occurs in adults. First, we quantified LsoA and LsoB titers in the guts of third- and fifth-instar potato psyllid nymphs. We found similar LsoA titers in the two nymphal stages, while LsoB titer was lower in the gut of the third-instar nymphs compared to fifth-instar nymphs. Second, we assessed the transmission efficiency of LsoA and LsoB by third-instar nymphs to tomato plants, revealing that LsoA was transmitted earlier and with higher efficiency than LsoB. Finally, we examined the transmission of LsoD by carrot psyllid nymphs to celery plants and demonstrated an age-related difference in the transmission rate. These findings provide valuable insights into the transmission dynamics of different Lso haplotypes by nymphal vectors, shedding light on their epidemiology and interactions with their psyllid vectors.

The plant pathogenic bacterium Candidatus Liberibacter solanacearum induces calcium-regulated autophagy in midgut cells of its insect vector Bactericera trigonica.

Autophagy plays an important role against pathogen infection in many organisms; however, little has been done with regard to vector-borne plant and animal pathogens, that sometimes replicate and cause deleterious effects in their vectors. Candidatus Liberibacter solanacearum (CLso) is a fastidious gram-negative phloem-restricted plant pathogen and vectored by the carrot psyllid, Bactericera trigonica. The plant disease caused by this bacterium is called carrot yellows and has recently gained much importance due to worldwide excessive economical losses. Here, we demonstrate that calcium ATPase, cytosolic calcium, and most importantly Beclin-1 have a role in regulating autophagy and its association with Liberibacter inside the psyllid. The presence of CLso generates reactive oxygen species and induces the expression of detoxification enzymes in the psyllid midguts, a main site for bacteria transmission. CLso also induces the expression of both sarco/endoplasmic reticulum Ca2+pump (SERCA) and 1,4,5-trisphosphate receptors (ITPR) in midguts, resulting in high levels of calcium in the cellular cytosol. Silencing these genes individually disrupted the calcium levels in the cytosol and resulted in direct effects on autophagy and subsequently on Liberibacter persistence and transmission. Inhibiting Beclin1-phosphorylation through different calcium-induced kinases altered the expression of autophagy and CLso titers and persistence. Based on our results obtained from the midgut, we suggest the existence of a direct correlation between cytosolic calcium levels, autophagy, and CLso persistence and transmission by the carrot psyllid. IMPORTANCE Plant diseases caused by vector-borne Liberibacter species are responsible for the most important economic losses in many agricultural sectors. Preventing these diseases relies mostly on chemical sprays against the insect vectors. Knowledge-based interference with the bacteria-vector interaction remains a promising approach as a sustainable solution. For unravelling how Liberibacter exploits molecular pathways in its insect vector for transmission, here, we show that the bacterium manipulates calcium levels on both sides of the endoplasmic reticulum membrane, resulting in manipulating autophagy. Silencing genes associated with these pathways disrupted the calcium levels in the cytosol and resulted in direct effects on autophagy and Liberibacter transmission. These results demonstrate major pathways that could be exploited for manipulating and controlling the disease transmission.

Association of endoplasmic reticulum associated degradation (ERAD) with the transmission of Liberibacter solanacearum by its psyllid vector.

Candidatus Liberibacter solanacearum (CLso) is a complex of gram negative plant pathogenic and fastidious bacterial haplotypes restricted to the phloem and transmitted by several psyllid species. In Israel, the carrot psyllid Bactericera trigonica transmits CLso haplotype D in a persistent and propagative manner and causes the carrot yellows disease, inflicting significant economic losses in many countries. Understanding the transmission of CLso is fundamental to devising sustainable management strategies. Persistent transmission of vector-borne pathogens involves the critical steps of adhesion, cell invasion and replication inside the insect gut cells before passage to the hemolymph. Using microscopy and expression analyses, we have previously confirmed a role for the endoplasmic reticulum (ER) in inducing immune responses and subsequent molecular pathways resulting in programmed cell death (apoptosis) upon CLso-infection in the midgut. In the current study, we confirm that the ER-associated degradation (ERAD) machinery and its associated marker genes were upregulated in CLso infected insects, including Derlin-1, Selenoprotein-1 and Ubiquitin Ligase RNF-185. Silencing Derlin-1, which acts on the ER membrane by regulating the degradation of unfolded proteins upon ER stress, revealed its role in CLso persistence and transmission. Molecular pathways initiated in the ER membrane upon bacterial infection are well documented in human, animal and insect systems, and this study confirms the role of the ER in CLso-psyllid interactions.

A C2H2 zinc finger transcription factor of the whitefly Bemisia tabaci interacts with the capsid proteins of begomoviruses and inhibits virus retention.

Begomoviruses are a group of ssDNA viruses exclusively transmitted by the whitefly Bemisia tabaci and constrain vegetable production in the old and new worlds. Although multiple molecular determinants governing the transmission of begomoviruses by whiteflies have been unravelled, factors critical for transmission majorly remain unknown. In this study, a whitefly C2H2 zinc finger (ZF) protein, 100% identical to the vascular endothelial ZF-like gene (vezf) protein was confirmed to interact with the CP of both old- and new-world begomoviruses. This was achieved by a yeast two-hybrid (Y2H) system screening of a whitefly cDNA library using capsid protein (CP) of TYLCV as a bait. In silico annotation of vezf protein revealed that it contains a N-terminal ZF-associated domain (ZAD) alongside multiple C2H2 ZF domains on the C-terminal end. ZAD-ZF proteins form the most abundant class of transcription factors within insects. Herein, we validated the interaction of vezf with four diverse begomoviruses and its functional role in begomovirus transmission. Silencing of the vezf gene of B. tabaci led to increased retention of three diverse begomoviruses tested. Vezf is the first insect transcription factor identified to interact with plant viruses and can be crucial to understand the possible mechanisms by which plant viruses modulate transcription of their insect vectors during transmission.

Comparative Analysis of Volatiles Emitted from Tomato and Pepper Plants in Response to Infection by Two Whitefly-Transmitted Persistent Viruses.

The whitefly Bemisia tabaci is one of the most important agricultural pests due to its extreme invasiveness, insecticide resistance, and ability to transmit hundreds of plant viruses. Among these, Begomoviruses and recombinant whitefly-borne Poleroviruses are transmitted persistently. Several studies have shown that upon infection, plant viruses manipulate plant-emitted volatile organic compounds (VOCs), which have important roles in communication with insects. In this study, we profiled and compared the VOCs emitted by tomato and pepper plant leaves after infection with the Tomato yellow leaf curl virus (TYLCV) (Bogomoviruses) and the newly discovered Pepper whitefly-borne vein yellows virus (PeWBVYV) (Poleroviruses), respectively. The results identified shared emitted VOCs but also uncovered unique VOC signatures for each virus and for whitefly infestation (i.e., without virus infection) independently. The results suggest that plants have general defense responses; however, they are also able to respond individually to infection with specific viruses or infestation with an insect pest. The results are important to enhance our understanding of virus- and insect vector-induced alteration in the emission of plant VOCs. These volatiles can eventually be used for the management of virus diseases/insect vectors by either monitoring or disrupting insect-plant interactions.

The SWC4 subunit of the SWR1 chromatin remodeling complex is involved in varying virulence of Metarhizium brunneum isolates offering role of epigenetic regulation of pathogenicity.

The host - pathogen interaction is a multifactorial process subject to a co-evolutionary arms race consisting of rapid changes in both host and pathogen, controlled at the genetic and epigenetic levels. Previously, we showed intra-species variation in disease progression and pathogenicity in aphids for Metarhizium brunneum isolates MbK and Mb7. Herein, we compared genomic, epigenetic, and metabolomic variations between these isolates and their effects on pathogenicity. Genomic variation could not completely explain the observed differences between the isolates. However, differential N6-adenine methylation (6 mA) and its correlation to reduced expression of the essential SWC4 subunit of SWR1 chromatin-remodelling complex (SWR1-C) led us to hypothesize a role for swc4 in the varying pathogenicity. Mutagenesis of the essential swc4 gene in MbKisolate resulted in reduction of secondary-metabolite (SM) secretion and impaired virulence in Galleria mellonella. Our results suggest the role of SWC4 in the regulation of SMs and the role of both SWC4 and SWR1-C in virulence of M. brunneum isolates. A better understanding of epigenetic regulation of SM production and secretion in entomopathogenic fungi may enable theirmanipulation for better biocontrol performance, and expand possibilities for environmentally friendly pest control.

Interaction of Liberibacter Solanacearum with Host Psyllid Vitellogenin and Its Association with Autophagy.

Candidatus Liberibacter solanacearum (CLso) haplotype D, transmitted by the carrot psyllid Bactericera trigonica, is a major constraint for carrot production in Israel. Unveiling the molecular interactions between the psyllid vector and CLso can facilitate the development of nonchemical approaches for controlling the disease caused by CLso. Bacterial surface proteins are often known to be involved in adhesion and virulence; however, interactions of CLso with carrot psyllid proteins that have a role in the transmission process has remained unexplored. In this study, we used CLso outer membrane protein (OmpA) and flagellin as baits to screen for psyllid interacting proteins in a yeast two-hybrid system assay. We identified psyllid vitellogenin (Vg) to interact with both OmpA and flagellin of CLso. As Vg and autophagy are often tightly linked, we also studied the expression of autophagy-related genes to further elucidate this interaction. We used the juvenile hormone (JH-III) to induce the expression of Vg, thapsigargin for suppressing autophagy, and rapamycin for inducing autophagy. The results revealed that Vg negatively regulates autophagy. Induced Vg expression significantly suppressed autophagy-related gene expression and the levels of CLso significantly increased, resulting in a significant mortality of the insect. Although the specific role of Vg remains obscure, the findings presented here identify Vg as an important component in the insect immune responses against CLso and may help in understanding the initial molecular response in the vector against Liberibacter. IMPORTANCE Pathogen transmission by vectors involves multiple levels of interactions, and for the transmission of liberibacter species by psyllid vectors, much of these interactions are yet to be explored. Candidatus Liberibacter solanacearum (CLso) haplotype D inflicts severe economic losses to the carrot industry. Understanding the specific interactions at different stages of infection is hence fundamental and could lead to the development of better management strategies to disrupt the transmission of the bacteria to new host plants. Here, we show that two liberibacter membrane proteins interact with psyllid vitellogenin and also induce autophagy. Altering vitellogenin expression directly influences autophagy and CLso abundance in the psyllid vector. Although the exact mechanism underlying this interaction remains unclear, this study highlights the importance of immune responses in the transmission of this disease agent.

Interactions of Liberibacter Species with Their Psyllid Vectors: Molecular, Biological and Behavioural Mechanisms.

Liberibacter is a group of plant pathogenic bacteria, transmitted by insect vectors, psyllids (Hemiptera: Psylloidea), and has emerged as one of the most devastating pathogens which have penetrated into many parts of the world over the last 20 years. The pathogens are known to cause plant diseases, such as Huanglongbing (citrus greening disease), Zebra chip disease, and carrot yellowing, etc., threatening some very important agricultural sectors, including citrus, potato and others. Candidatus Liberibacter asiaticus (CLas), the causative agent of citrus greening disease, is one of the most important pathogens of this group. This pathogen has infected most of the citrus trees in the US, Brazil and China, causing tremendous decline in citrus productivity, and, consequently, a severely negative impact on economic and personnel associated with citrus and related industries in these countries. Like other members in this group, CLas is transmitted by the Asian citrus psyllid (ACP, Diaphorina citri) in a persistent circulative manner. An additional important member of this group is Ca. L. solanacearum (CLso), which possesses nine haplotypes and infects a variety of crops, depending on the specific haplotype and the insect vector species. Ongoing pathogen control strategies, that are mainly based on use of chemical pesticides, lack the necessary credentials of being technically feasible, and environmentally safe. For this reason, strategies based on interference with Liberibacter vector transmission have been adopted as alternative strategies for the prevention of infection by these pathogens. A significant amount of research has been conducted during the last 10-15 years to understand the aspects of transmission of these bacterial species by their psyllid vectors. These research efforts span biological, ecological, behavioural and molecular aspects of Liberibacter-psyllid interactions, and will be reviewed in this manuscript. These attempts directed towards devising new means of disease control, endeavoured to explore alternative strategies, instead of relying on using chemicals for reducing the vector populations, which is the sole strategy currently employed and which has profound negative effects on human health, beneficial organisms and the environment.