Validation of ‘Variable Number of Tandem Repeat’-Based Approach for Examination of ‘Candidatus Liberibacter asiaticus’ Diversity and Its Applications for the Analysis of the Pathogen Populations in the Areas of Recent Introduction

Citrus greening (Huanglongbing, HLB) is one of the most destructive diseases of citrus worldwide. In South Asia HLB has been known for more than a century, while in Americas the disease was found relatively recently. HLB is associated with three species of ‘Candidatus Liberibacter’ among which ‘Ca. Liberibacter asiaticus’ (CLas) has most wide distribution. Recently, a number of studies identified different regions in the CLas genome with variable number of tandem repeats (VNTRs) that could be used for examination of CLas diversity. One of the objectives of the work presented here was to further validate the VNTR analysis-based approach by assessing the stability of these repeats upon multiplication of the pathogen in a host over an extended period of time and upon its passaging from a host to a host using CLas populations from Florida. Our results showed that the numbers of tandem repeats in the four loci tested display very distinguishable “signature profiles” for the two Florida-type CLas haplotype groups. Remarkably, the profiles do not change upon passage of the pathogen in citrus and psyllid hosts as well as after its presence within a host over a period of five years, suggesting that VNTR analysis-based approach represents a valid methodology for examination of the pathogen populations in various geographical regions. Interestingly, an extended analysis of CLas populations in different locations throughout Florida and in several countries in the Caribbean and Central America regions and in Mexico where the pathogen has been introduced recently demonstrated the dispersion of the same haplotypes of CLas. On the other hand, these CLas populations appeared to differ significantly from those obtained from locations where the disease has been present for a much longer time.     

Infection with Strains of Citrus Tristeza Virus Does Not Exclude Superinfection by Other Strains of the Virus

Superinfection exclusion or homologous interference, a phenomenon in which a primary viral infection prevents a secondary infection with the same or closely related virus, has been observed commonly for viruses in various systems, including viruses of bacteria, plants, and animals. With plant viruses, homologous interference initially was used as a test of virus relatedness to define whether two virus isolates were “strains” of the same virus or represented different viruses, and subsequently purposeful infection with a mild isolate was implemented as a protective measure against isolates of the virus causing severe disease. In this study we examined superinfection exclusion of Citrus tristeza virus (CTV), a positive-sense RNA closterovirus. Thirteen naturally occurring isolates of CTV representing five different virus strains and a set of isolates originated from virus constructs engineered based on an infectious cDNA clone of T36 isolate of CTV, including hybrids containing sequences from different isolates, were examined for their ability to prevent superinfection by another isolate of the virus. We show that superinfection exclusion occurred only between isolates of the same strain and not between isolates of different strains. When isolates of the same strain were used for sequential plant inoculation, the primary infection provided complete exclusion of the challenge isolate, whereas isolates from heterologous strains appeared to have no effect on replication, movement or systemic infection by the challenge virus. Surprisingly, substitution of extended cognate sequences from isolates of the T68 or T30 strains into T36 did not confer the ability of resulting hybrid viruses to exclude superinfection by those donor strains. Overall, these results do not appear to be explained by mechanisms proposed previously for other viruses. Moreover, these observations bring an understanding of some previously unexplained fundamental features of CTV biology and, most importantly, build a foundation for the strategy of selecting mild isolates that would efficiently exclude severe virus isolates as a practical means to control CTV diseases.Superinfection exclusion or homologous interference is a phenomenon in which a preexisting viral infection prevents a secondary infection with the same or a closely related virus, whereas infection by unrelated viruses can be unaffected. The phenomenon was first observed by McKinney (57, 58) between two genotypes of Tobacco mosaic virus (TMV) and later with bacteriophages (21, 94). Since that time, the phenomenon has been observed often for viruses of animals (1, 13, 18, 34, 43, 47, 50, 85, 86-88, 102, 103) and plants (11, 30, 31, 32, 39, 40, 49, 77, 99, 100). In plant virology, homologous interference initially was used as a test of virus relatedness to define whether two virus isolates were “strains” of the same virus or represented different viruses (58, 77). Subsequently, it was developed into a management tool to reduce crop losses by purposely infecting plants with mild isolates of a virus to reduce infection and losses due to more severe isolates, which is referred to as “cross-protection” (reviewed in references 32 and 40).Homologous superinfection exclusion of animal viruses has been related to several mechanisms acting at various stages of the viral life cycle, including prevention of the incoming virus entry into cells (50, 86, 87), or inhibition of translation or interference with replication (1, 47, 50, 83). Several mechanisms have been postulated for homologous interference of plant viruses, including prevention of the disassembly of the challenge virus as it enters the cell resulting from the expression of the coat protein of the protector virus (67, 84; reviewed in reference 10) and induction of RNA silencing by the protector virus that leads to sequence-specific degradation of the challenge virus RNA (24, 69, 70). However, common mechanisms of superinfection exclusion, expected to be associated with the viruses of plants and animals, have not been elucidated.Citrus tristeza virus (CTV) is the largest and most complex member of the Closteroviridae family, which contains viruses with mono-, bi-, and tripartite genomes transmitted by a range of insect vectors, including aphids, whiteflies, and mealybugs (3, 6, 19, 20, 46). CTV has long flexuous virions (2,000 nm by 10 to 12 nm) encapsidated by two coat proteins and a single-stranded RNA genome of ∼19.3 kb. The major coat protein (CP) covers ca. 97% of the genomic RNA, and the minor coat protein (CPm) completes encapsidation of the genome at its 5′ end (25, 81). The RNA genome of CTV encodes 12 open reading frames (ORFs) (44, 64) (Fig. ​(Fig.1).1). ORFs 1a and 1b are expressed from the genomic RNA and encode polyproteins required for virus replication. ORF 1a encodes a 349-kDa polyprotein containing two papainlike protease domains plus methyltransferaselike and helicaselike domains. Translation of the polyprotein is thought to occasionally continue through the polymerase-like domain (ORF 1b) by a +1 frameshift. Ten 3′-end ORFs are expressed by 3′-coterminal subgenomic RNAs (sgRNAs) (37, 45) and encode the following proteins: major (CP) and minor (CPm) coat proteins, p65 (HSP70 homolog), and p61 that are involved in assembly of virions (79); a hydrophobic p6 protein with a proposed role in virus movement (20, 89); p20 and p23, which along with CP are suppressors of RNA silencing (54); and p33, p13, and p18, whose functions remain unknown. Remarkably, citrus trees can be infected with mutants with three genes deleted: p33, p18, and p13 (89).Open in a separate windowFIG. 1.(A) Schematic diagram of the genome organization of wild-type CTV (CTV9R) and its derivative CTV-BC5/GFP encoding GFP. The open boxes represent ORFs and their translation products. PRO, papainlike protease domain; MT, methyltransferase; HEL, helicase; RdRp, an RNA-dependent RNA polymerase; HSP70h, HSP70 homolog; CPm, minor coat protein; CP, major coat protein; GFP, green fluorescent protein. Bent arrows indicate positions of BYV (B_CP) or CTV CP (C_CP) sgRNA controller elements. Inserted elements are shown in gray. (B) Scheme of the “superinfection exclusion assay.” Young Madam Vinous sweet orange trees were initially inoculated with one of 13 tested CTV isolates. When primary infections were established, the trees were subsequently challenged with CTV-BC5/GFP. All inoculations were done by grafting of the infected tissue into the stem of a tree. The positions of primary (Pri) and challenge (Chl) graft inoculations are shown. The ability of the challenge virus to superinfect trees was determined by visual observation of GFP fluorescence in phloem-associated cells on the internal surface of bark from a young flash starting at about 2 months upon challenge inoculation. Scale bar, 0.4 mm.The host range of CTV is limited to citrus in which the virus infects only phloem-associated cells. CTV consists of numerous isolates that have distinctive biological and genetic characteristics (38, 48, 56, 72, 74, 75, 95). Recently, a classification strategy for CTV isolates was proposed based on sequence similarity. Analysis of nearly 400 isolates in an international collection revealed five major CTV genotype groups with some isolates undefined (38). For the purposes of the present study, strains are defined as phylogenetically distinct lineages of CTV based upon analysis of nucleotide sequences of the 1a ORF (38). This region of the genome shows high genetic diversity between CTV variants, with levels of sequence identity ranging between 72.3 to 90.3% (38, 48, 52, 74, 75; M. Hilf, unpublished data). Using this definition, T3, T30, T36, VT, and T68 are designated as strains. Individual virus samples are designated as isolates of one of these strains. The ORF 1a nucleotide sequences of isolates of the T36 and T68 strains are equally dissimilar to isolates of the T3, T30, and VT strains, with identities of 72.9, 73, and 72.4% and 77.6, 77.9, and 76.8%, respectively. Identities of ORF 1a range from 89.4 to 90.3% between isolates of the T3, T30, and VT strains. Sequences of ORF1a of isolates belonging to the T36 strain and those from the T68 strain show 72.3% identity. This compares to a range of 89 to 94.8% identity found in the more conserved 3′-half regions of the genomes of isolates from different CTV strains. Each strain is named after a “type isolate” and is composed of isolates with minor sequence divergence (generally less than 5% throughout genome) from the type member. However, isolates of a strain may have significant variations in symptoms and symptoms severity. Remarkably, field trees harbor complex populations of CTV, which are often composed of mixtures of different strains and recombinants between these strains (36, 48, 52, 68, 75, 96, 101). The genetic basis of such frequent coexistence of different strains within the same tree is unknown.CTV causes economically important diseases of citrus worldwide. One of the most effective management tools has been cross-protection when effective protecting isolates could be found. Preinfection with mild isolates allows commercial production of sweet oranges and limes in Brazil (16) and Peru (9) and grapefruit in South Africa (92). However, identification of protecting isolates has been empirical, difficult, and rare. Cross-protection usually has worked only in certain varieties, and the lack of effective protecting isolates has prevented its use in many varieties and citrus growing areas (15, 41, 61, 73). In general, there has been no understanding why some mild isolates were effective and others failed to protect. Because CTV diseases prevail in citrus growing areas worldwide, elucidation of the mechanisms of exclusion of one CTV variant by another one is an important goal.In the present study we examined relationships between different genotypes of CTV in terms of their ability to prevent superinfection by another isolate of the virus. We show that superinfection exclusion occurred only between minor genetic variants of the same strain (sequence group) and not between isolates of different strains. When isolates of the same strain were used for sequential plant inoculation, the primary infection provided full exclusion of the challenge isolate. In all combinations of virus isolates belonging to different strains, the primary infection of plants with one strain had no noticeable effect on the establishment of the secondary infection. The results obtained here help elucidate some previously unexplained fundamental features of CTV biology and pose the possibility of an existence of a novel mechanism for superinfection exclusion between virus variants.     

Citrus tristeza virus: survival at the edge of the movement continuum

Systemic invasion of plants by viruses is thought to involve two processes: cell-to-cell movement between adjacent cells and long-distance movement that allows the virus to rapidly move through sieve elements and unload at the growing parts of the plant. There is a continuum of proportions of these processes that determines the degrees of systemic infection of different plants by different viruses. We examined the systemic distribution of Citrus tristeza virus (CTV) in citrus species with a range of susceptibilities. By using a "pure" culture of CTV from a cDNA clone and green fluorescent protein-labeled virus we show that both cell-to-cell and long-distance movement are unusually limited, and the degree of limitation varies depending on the citrus host. In the more-susceptible hosts CTV infected only a small portion of phloem-associated cells, and moreover, the number of infection sites in less-susceptible citrus species was substantially decreased further, indicating that long-distance movement was reduced in those hosts. Analysis of infection foci in the two most differential citrus species, Citrus macrophylla and sour orange, revealed that in the more-susceptible host the infection foci were composed of a cluster of multiple cells, while in the less-susceptible host infection foci were usually single cells, suggesting that essentially no cell-to-cell movement occurred in the latter host. Thus, CTV in sour orange represents a pattern of systemic infection in which the virus appears to function with only the long-distance movement mechanism, yet is able to survive in nature.     

Activation of hypothalamic RIP‐Cre neurons promotes beiging of WAT via sympathetic nervous system

Activation of brown adipose tissue (BAT) and beige fat by cold increases energy expenditure. Although their activation is known to be differentially regulated in part by hypothalamus, the underlying neural pathways and populations remain poorly characterized. Here, we show that activation of rat‐insulin‐promoter‐Cre (RIP‐Cre) neurons in ventromedial hypothalamus (VMH) preferentially promotes recruitment of beige fat via a selective control of sympathetic nervous system (SNS) outflow to subcutaneous white adipose tissue (sWAT), but has no effect on BAT. Genetic ablation of APPL2 in RIP‐Cre neurons diminishes beiging in sWAT without affecting BAT, leading to cold intolerance and obesity in mice. Such defects are reversed by activation of RIP‐Cre neurons, inactivation of VMH AMPK, or treatment with a β3‐adrenergic receptor agonist. Hypothalamic APPL2 enhances neuronal activation in VMH RIP‐Cre neurons and raphe pallidus, thereby eliciting SNS outflow to sWAT and subsequent beiging. These data suggest that beige fat can be selectively activated by VMH RIP‐Cre neurons, in which the APPL2–AMPK signaling axis is crucial for this defending mechanism to cold and obesity.     

Proteomic analysis during larval development and metamorphosis of the spionid polychaete Pseudopolydora vexillosa

Background  

While the larval-juvenile transition (metamorphosis) in the spionid polychaete Pseudopolydora vexillosa involves gradual morphological changes and does not require substantial development of juvenile organs, the opposite occurs in the barnacle Balanus amphitrite. We hypothesized that the proteome changes during metamorphosis in the spionids are less drastic than that in the barnacles. To test this, proteomes of pre-competent larvae, competent larvae (ready to metamorphose), and juveniles of P. vexillosa were compared using 2-dimensional gel electrophoresis (2-DE), and they were then compared to those of the barnacle.