Zhongguo Xiong

Extracellular DNA Is Required for Root Tip Resistance to Fungal Infection

Plant defense involves a complex array of biochemical interactions, many of which occur in the extracellular environment. The apical 1- to 2-mm root tip housing apical and root cap meristems is resistant to infection by most pathogens, so growth and gravity sensing often proceed normally even when other sites on the root are invaded. The mechanism of this resistance is unknown but appears to involve a mucilaginous matrix or “slime” composed of proteins, polysaccharides, and detached living cells called “border cells.” Here, we report that extracellular DNA (exDNA) is a component of root cap slime and that exDNA degradation during inoculation by a fungal pathogen results in loss of root tip resistance to infection. Most root tips (>95%) escape infection even when immersed in inoculum from the root-rotting pathogen Nectria haematococca. By contrast, 100% of inoculated root tips treated with DNase I developed necrosis. Treatment with BAL31, an exonuclease that digests DNA more slowly than DNase I, also resulted in increased root tip infection, but the onset of infection was delayed. Control root tips or fungal spores treated with nuclease alone exhibited normal morphology and growth. Pea (Pisum sativum) root tips incubated with [32P]dCTP during a 1-h period when no cell death occurs yielded root cap slime containing 32P-labeled exDNA. Our results suggest that exDNA is a previously unrecognized component of plant defense, an observation that is in accordance with the recent discovery that exDNA from white blood cells plays a key role in the vertebrate immune response against microbial pathogens.

Synthesis of the putative red clover necrotic mosaic virus RNA polymerase by ribosomal frameshifting in vitro.

Abstract The red clover necrotic mosaic virus (RCNMV) genome is split between two single-stranded RNA species termed RNA-1 and RNA-2. RNA-1 directs the synthesis of 88-kDa (p88), 57-kDa (p57), 37-kDa (p37), and 27-kDa (p27) polypeptides and RNA-2 a 35-kDa (p35) polypeptide in vitro. The coding order of the RNA-1 products was determined to be 5′-p27-p57-p37-3′. Antibodies to synthetic peptides representing the carboxyl terminal portions of p27 and p57 immunoprecipitated their respective polypeptides in addition to p88, suggesting that p88 is a fusion protein. A frameshift heptanucleotide sequence element has been identified in RCNMV RNA-1. In addition, a stable stem-loop secondary structure adjacent to the heptanucleotide sequence is predicted. Together, these sequence elements suggest that a ribosomal frameshifting event occurs which allows translational readthrough of the p27 open reading frame into the p57 open reading frame, generating the observed p88 product. An RNA-1 expression construct fusing the p57 and the CP open reading frame was engineered to investigate the ribosomal frameshifting event. CP antibodies immunoprecipitated a fusion protein of the predicted size containing the carboxyl portion of CP. Site-directed mutagenesis of the frameshift element indicates that in vitro, p88 can also be expressed alternatively by suppression of an amber termination codon. Based on these data, we propose that the putative RCNMV RNA polymerase is an 88-kDa polypeptide expressed by a ribosomal frameshifting mechanism similar to those utilized by retroviruses.

Advances in plant virus evolution: Translating evolutionary insights into better disease management

Recent studies in plant virus evolution are revealing that genetic structure and behavior of virus and viroid populations can explain important pathogenic properties of these agents, such as host resistance breakdown, disease severity, and host shifting, among others. Genetic variation is essential for the survival of organisms. The exploration of how these subcellular parasites generate and maintain a certain frequency of mutations at the intra- and inter-host levels is revealing novel molecular virus-plant interactions. They emphasize the role of host environment in the dynamic genetic composition of virus populations. Functional genomics has identified host factors that are transcriptionally altered after virus infections. The analyses of these data by means of systems biology approaches are uncovering critical plant genes specifically targeted by viruses during host adaptation. Also, a next-generation resequencing approach of a whole virus genome is opening new avenues to study virus recombination and the relationships between intra-host virus composition and pathogenesis. Altogether, the analyzed data indicate that systematic disruption of some specific parameters of evolving virus populations could lead to more efficient ways of disease prevention, eradication, or tolerable virus-plant coexistence.

Persistent Infection and Promiscuous Recombination of Multiple Genotypes of an RNA Virus within a Single Host Generate Extensive Diversity

Recombination and reassortment of viral genomes are major processes contributing to the creation of new, emerging viruses. These processes are especially significant in long-term persistent infections where multiple viral genotypes co-replicate in a single host, generating abundant genotypic variants, some of which may possess novel host-colonizing and pathogenicity traits. In some plants, successive vegetative propagation of infected tissues and introduction of new genotypes of a virus by vector transmission allows for viral populations to increase in complexity for hundreds of years allowing co-replication and subsequent recombination of the multiple viral genotypes. Using a resequencing microarray, we examined a persistent infection by a Citrus tristeza virus (CTV) complex in citrus, a vegetatively propagated, globally important fruit crop, and found that the complex comprised three major and a number of minor genotypes. Subsequent deep sequencing analysis of the viral population confirmed the presence of the three major CTV genotypes and, in addition, revealed that the minor genotypes consisted of an extraordinarily large number of genetic variants generated by promiscuous recombination between the major genotypes. Further analysis provided evidence that some of the recombinants underwent subsequent divergence, further increasing the genotypic complexity. These data demonstrate that persistent infection of multiple viral genotypes within a host organism is sufficient to drive the large-scale production of viral genetic variants that may evolve into new and emerging viruses.

Roles of root border cells in plant defense and regulation of rhizosphere microbial populations by extracellular DNA 'trapping'

BackgroundAs roots penetrate soil, specialized cells called ‘border cells’ separate from root caps and contribute a large proportion of exudates forming the rhizosphere. Their function has been unclear. Recent findings suggest that border cells act in a manner similar to that of white blood cells functioning in defense. Histone-linked extracellular DNA (exDNA) and proteins operate as ‘neutrophil extracellular traps’ to attract and immobilize animal pathogens. DNase treatment reverses trapping and impairs defense, and mutation of pathogen DNase results in loss of virulence.ScopeHistones are among a group of proteins secreted from living border cells. This observation led to the discovery that exDNA also functions in defense of root caps. Experiments revealed that exDNA is synthesized and exported into the surrounding mucilage which attracts, traps and immobilizes pathogens in a host-microbe specific manner. When this plant exDNA is degraded, the normal resistance of the root cap to infection is abolished.ConclusionsResearch to define how exDNA may operate in plant immunity is needed. In the meantime, the specificity and stability of exDNA and its association with distinct microbial species may provide an important new tool to monitor when, where, and how soil microbial populations become established as rhizosphere communities.

Complementary functions of two recessive R-genes determine resistance durability of tobacco 'Virgin A Mutant' (VAM) to Potato virus Y.

Tobaccos VAM and NC745 carry the recessive va gene that confers resistance to PVY(NN). However, they exhibit different levels of resistance durability. Upon virus inoculation, only NC745 developed sporadic systemic symptoms caused by emerging resistance-breaking variants that easily infected both NC745 and VAM genotypes. To identify the differential host conditions associated with this phenomenon, cellular accumulation, cell-to-cell movement, vascular translocation, and foliar content of PVY(NN) were comparatively evaluated. Virus cell-to-cell movement was restricted and its transit through the vasculature boundaries was completely blocked in both tobacco varieties. However, an additional defense mechanism operating only in tobacco VAM drastically reduced the in situ cellular virus accumulation. Genetic analyses of hybrid plant progenies indicate that VAM-type resistance was conditioned by at least two recessive genes: va and a newly reported va2 locus. Moreover, segregant plant progenies that restricted virus movement but permitted normal virus accumulation were prone to develop resistance-breaking infections.

Paternal inheritance of chloroplast DNA in interspecific hybrids in the genus Larrea (Zygophyllaceae)

The mode of chloroplast DNA (cpDNA) inheritance was investigated in the genus Larrea (Zygophyllaceae) by polymerase chain reaction (PCR) amplification of cpDNA fragments using three pairs of chloroplast universal primers. A total of 20 F(1)s from interspecific crosses among five different taxa in the section Bifolium was examined. Twelve F(1)s were from six crosses between L. cuneifolia (4x) and L. divaricata (2x) (Peru or Argentina) or L. tridentata (2x or 4x). Eight F(1)s were from two sets of reciprocal crosses between L. divaricata (2x) (Argentina) and L. tridentata (2x). Length polymorphism was observed in all three regions of cpDNA that separated L. cuneifolia parents from L. divaricata and L. tridentata parents and in one of the three cpDNA regions that differentiated L. divaricata (Argentina) parents from L. tridentata (2x) parents. In each case, it was the paternal cpDNA marker that appeared in the F(1) individuals. This was further confirmed by restriction fragment length polymorphism (RFLP) analysis of the amplified cpDNA fragments. Larrea may be the fifth genus reported in angiosperms with a paternal bias in cpDNA transmission. Possible mechanisms that may result in paternal cpDNA inheritance were briefly reviewed. Based on the observed uniparental paternal inheritance of cpDNA, restriction analysis of the three cpDNA regions and previous cytogenetic studies, L. divaricata was probably the maternal progenitor of L. cuneifolia.

Nucleotide sequence of the capsid protein cistrons from six potato virus Y (PVY) isolates infecting tobacco.

SummaryComplementary DNA libraries representing the capsid protein cistron of the potato virus Y (PVY) isolate ‘Chilean’, ‘Hungarian’, MsNr, NsNr, O, and ‘Potato US’ were synthesized and used as template for polymerase chain reaction (PCR) amplification. An AUG codon for initiating a discrete capsid protein (CP) open reading frame was embedded upstream of the first codon of the CP cistrons. PCR-amplified products of the expected size of 0.8 kilo bases were cloned into the transcription vector pBS(+). The fidelity of each PCR-amplified PVY CP cistron was tested by transcribing recombinant plasmids in vitro and translating the transcripts in two cell free translation systems. Translation analysis of in vitro transcribed PVY CP cistrons consistently yielded a polypeptide co-migrating with authentic CP that was immunoprecipitated by anti PVY ‘Chilean’ antibodies. The nucleotide sequence of each capsid protein gene was determined by dideoxy sequence analysis. Each capsid protein gene was determined to be 801 nucleotides in length, encoding a deduced protein of 267 amino acids with calculated Mr ranging from 29 799 to 29 980. The nucleic acid sequence similarity between the six isolates ranged between 89 to 97% and the amino acid similarity between 91 to 99%. The high level of amino acid sequence similarity confirms the classification of these viruses as isolates of PVY.

Complete genomic sequence of a Papaya ringspot virus isolate from Hainan Island, China

Papaya ringspot virus (PRSV, genus Potyvirus, family Potyviridae) causes significant yield losses in papaya and in some cucurbits throughout tropical and subtropical regions [7]. Based on its host specificity, PRSV is classified into two biotypes. Type W is known to infect only cucurbits, whereas type P infects both papaya and cucurbits. The first PRSV genome completely sequenced and characterized was the Hawaiian isolate, PRSV-H [11]. The complete sequences of a number of PRSV isolates have since been determined [2, 3, 5, 6, 10]. Hainan Island, having 1,300 ha of papaya plantation and an annual output of 70,000 tons, is the most important papaya-producing region in China. The papaya ringspot disease has been and will remain a major challenge in papaya production in Hainan. The disease in Hainan displayed typical symptoms of PRSV infection [11]. However, the causative virus and its relationship with other PRSV isolates have not been characterized. Understanding the exact nature of the PRSV in Hainan is important for the management of the disease. This importance is apparent in light of recent reports that described different specificities of PRSV resistance displayed by transgenic papaya lines expressing different PRSV CP genes toward PRSV isolates from different geographic origins [8, 9]. In this paper, we report the complete genomic sequence of PRSV (PRSVHN) from the Hainan Island, China, and present evidence that PRSV-HN represents a unique and novel PRSV isolate.

Intrahost mechanisms governing emergence of resistance-breaking variants of Potato virus Y.

The emergence of resistance breaking (RB) variants starting from the avirulent Potato virus Y NN strain (PVY(NN)) was analyzed after imposing different selective host constraints. Tobacco resistance to PVY(NN) is conferred by va in both NC745 and VAM genotypes, but VAM carries an extra resistance gene, va2. RB-variants emerged only in NC745 and unexpectedly accumulated higher in the original host, tobacco B21, than the parental PVY(NN). However, the recovery of RB-variants was interfered by PVY(NN) in mixed infections. Further analysis indicated that RB-variants also arose in tobacco VAM, but they were limited to subliminal local infections. Their inability to breakout was associated with absence of a mutational adaptation in the viral VPg gene, which implied a loss of fitness in tobacco B21. Altogether, the emergence of RB-variants was conditioned by inherited host constraints, interference by co-infecting avirulent virus genotypes, and fitness tradeoff of virus adaptations.