Functional analysis of Pcipg2 from the straminopilous plant pathogen Phytophthora capsici

Phytophthora capsici causes serious diseases in numerous crop plants. Polygalacturonases (PGs) are cell wall‐degrading enzymes that play an important role in pathogenesis in straminopilous pathogens. To understand PGs as they relate to the virulence of P. capsici, Pcipg2 was identified from a genomic library of a highly virulent P. capsici strain. Pcipg2 was strongly expressed during symptom development after the inoculation of pepper leaves with P. capsici. The wild protein (PCIPGII) was obtained from the expression of pcipg2 and found that increasing activity of PGs in PCIPGII‐treated pepper leaves was consistent with increasing symptom development. Asp residues in active sites within pcipg2 affected PCIPGII activity or its virulence on pepper leaves. Results show that pcipg2 is an important gene among pcipg genes, and illustrate the benefit of analyzing mechanisms of pathogenicity during the period of host/parasite interaction. genesis 47:535–544, 2009. © 2009 Wiley‐Liss, Inc.     

辣椒疫霉RXLR型效应子PcAvh2的序列多态性、基因转录特征及功能

【目的】分析辣椒疫霉中RXLR型效应子PcAvh2的序列多态性,研究该效应子在辣椒疫霉生长发育和侵染阶段的转录特征及其生物学功能。【方法】本研究通过高保真扩增,分析2个烟草疫霉、1个恶疫霉和31个辣椒疫霉菌株的PcAvh2序列;提取辣椒疫霉菌丝、游动孢子囊、游动孢子、萌发休止孢和7个侵染时间点(1.5、3、6、12、24、36、72 h)的本氏烟根部总RNA,利用RT-qPCR分析PcAvh2的转录表达水平;利用PVX瞬时表达系统,分析PcAvh2是否抑制6种效应子(BAX、INF1、PsojNIP、PsCRN63、PsAvh241、R3a/Avr3a)激发的植物免疫反应;利用CaCl_2-PEG介导的原生质体稳定转化技术,沉默PcAvh2基因,分析辣椒疫霉致病力的变化。【结果】PcAvh2为典型的RXLR效应子,在辣椒疫霉群体中该效应子具有10个等位基因,而且烟草疫霉和恶疫霉中也存在该效应子。该基因在辣椒疫霉的侵染阶段上调表达,它能够抑制6种效应子激发的植物免疫反应,进一步研究发现基因沉默导致辣椒疫霉的致病力显著下降。【结论】RXLR型效应子PcAvh2是辣椒疫霉中一个重要的侵染致病因子。     

编码腈水解酶的大豆疫霉基因PsNIA的克隆与转录分析

包括大豆在内的许多植物都可以产生氰化物,对侵染的病原菌产生毒害作用而阻碍其进一步扩展。采用抑制性差减杂交（suppression subtractive hybridization,SSH）的方法,筛选到一个在大豆疫霉侵染早期上调表达的、编码腈水解酶的cDNA片段;克隆了该基因的全长序列,命名为PsNIA。Southern杂交结果显示,PsNIA在大豆疫霉基因组中只有1个拷贝。系统发育分析表明,PsNIA与绿脓杆菌Pseudomonas aeruginosa的腈水解酶的序列同源性最高,且该基因编码的氨基酸序列具有腈水解酶的保守结构域。RT-PCR分析表明,该基因在大豆疫霉侵染大豆12h时可以检测到转录。     

编码腈水解酶的大豆疫霉基因PsNIA的克隆与转录分析

包括大豆在内的许多植物都可以产生氰化物,对侵染的病原菌产生毒害作用而阻碍其进一步扩展。采用抑制性差减杂交(suppression subtractive hybridization,SSH)的方法,筛选到一个在大豆疫霉侵染早期上调表达的、编码腈水解酶的cDNA片段;克隆了该基因的全长序列,命名为PsNIA。Southern杂交结果显示,PsNIA在大豆疫霉基因组中只有1个拷贝。系统发育分析表明,PsNIA与绿脓杆菌Pseudomonas aeruginosa的腈水解酶的序列同源性最高,且该基因编码的氨基酸序列具有腈水解酶的保守结构域。RT-PCR分析表明,该基因在大豆疫霉侵染大豆12h时可以检测到转录。     

Population genomics of an outbreak of the potato late blight pathogen,Phytophthora infestans,reveals both clonality and high genotypic diversity

An outbreak of the potato late blight pathogen Phytophthora infestans in Denmark was characterized in order to resolve the population structure and determine to what extent sexual reproduction was occurring. A standard set of microsatellite simple sequence repeats (SSRs) and single nucleotide polymorphism (SNP) markers generated using restriction site-associated DNA sequencing (RAD-seq) were employed in parallel. A total of 83 individuals, isolated from seven different potato fields in 2014, were analysed together with five Danish whole-genome sequenced isolates, as well as two Mexican individuals used as an outgroup. From a filtered dataset of 55 288 SNPs, population genomics analyses revealed no sign of recombination, implying clonality. In spite of this, multilocus genotypes were unique to individual potato fields, with little evidence of gene flow between fields. Ploidy analysis performed on the SNPs dataset indicated that the majority of isolates were diploid. These contradictory results with clonality and high genotypic diversity may suggest that rare sexual events likely still contribute to the population. Comparison of the results generated by SSRs vs SNPs data indicated that large marker sets, generated by RAD-seq, may be advised going forward, as it provides a higher level of genetic discrimination than SSRs.     

Trade‐offs and evolution of thermal adaptation in the Irish potato famine pathogen Phytophthora infestans

Temperature is one of the most important environmental parameters with crucial impacts on nearly all biological processes. Due to anthropogenic activity, average air temperatures are expected to increase by a few degrees in coming decades, accompanied by an increased occurrence of extreme temperature events. Such global trends are likely to have various major impacts on human society through their influence on natural ecosystems, food production and biotic interactions, including diseases. In this study, we used a combination of statistical genetics, experimental evolution and common garden experiments to investigate the evolutionary potential for thermal adaptation in the potato late blight pathogen, Phytophthora infestans, and infer its likely response to changing temperatures. We found a trade‐off associated with thermal adaptation to heterogeneous environments in P. infestans, with the degree of the trade‐off peaking approximately at the pathogen's optimum growth temperature. A genetic trade‐off in thermal adaptation was also evidenced by the negative association between a strain's growth rate and its thermal range for growth, and warm climates selecting for a low pathogen growth rate. We also found a mirror effect of phenotypic plasticity and genetic adaptation on growth rate. At below the optimum, phenotypic plasticity enhances pathogen's growth rate but nature selects for slower growing genotypes when temperature increases. At above the optimum, phenotypic plasticity reduces pathogen's growth rate but natural selection favours for faster growing genotypes when temperature increases further. We conclude from these findings that the growth rate of P. infestans will only be marginally affected by global warming.     

Reconstruction of the Sudden Oak Death epidemic in California through microsatellite analysis of the pathogen Phytophthora ramorum

The genetic structure of the clonally reproducing Sudden Oak Death (SOD) pathogen in California was investigated using seven variable microsatellites. A total of 35 multilocus genotypes were identified among 292 samples representative of populations from 14 forest sites and of the nursery trade. amova indicated significant genetic variability both within (44.34%) and among populations (55.66%). Spatial autocorrelation analyses indicated that Moran's index of similarity reached a minimum of 0.1 at 350 m, increased to 0.4 at 1500 m and then decreased to zero at 10 km. These results suggest a bimodal pattern of spread, with medium range dispersal (1500–10 000 m) putatively attributed to the presence of strong winds. Lack of genetic structure was identified for three groups of populations. One group notably included the nurseries' population and two forest populations, both linked to early reports of the pathogen. A neighbour-joining analysis based on pairwise Φ_ST values indicated that the clade inclusive of the nurseries' populations is basal to all California populations. A network analysis identified three common genotypes as the likely founders of the California infestation and proposes a stepwise model for local evolution of novel genotypes. This was supported by the identification in the same locations of novel genotypes and of their 1- or 2-step parents. We hypothesize that the few undifferentiated population groups indicate historical human spread of the pathogen, while the general presence of genetically structured populations indicates that new infestations are currently generated by rare medium or long-range natural movement of the pathogen, followed by local generation of new genotypes.     

Cloning and characterization of a theta class glutathione transferase from the potato pathogen Phytophthora infestans

A glutathione transferase (GST) related to the theta (T) class of enzymes found in plants and animals has been cloned from the potato pathogen Phytophthora infestans. The cDNA encoded a 25kDa polypeptide termed PiGSTT1 which was expressed in E. coli as the native protein. The purified recombinant enzyme behaved as a dimer (PiGSTT1-1) and while being unable to catalyse the glutathione conjugation of 1-chloro-2,4-dintrobenzene, was highly active as a glutathione peroxidase with organic hydroperoxide substrates. In addition to reducing the synthetic substrate cumene hydroperoxide, PiGSTT1-1 was shown to be highly active toward 9(S)-hydroperoxy-(10E,12Z,15Z)-octadecatrienoic acid=9(S)-HPOT, which is formed in potato plants during infection by P. infestans as a precursor of the antifungal oxylipin colnelenic acid. An antiserum was raised to PiGSTT1-1 and used to demonstrate that the respective enzyme was abundantly expressed in P. infestans both cultured on pea agar and during the infection of potato plants.     

Population dynamics of the sudden oak death pathogen Phytophthora ramorum in Oregon from 2001 to 2004

Phytophthora ramorum (Oomycetes) is an emerging plant pathogen in forests in southwestern Oregon (Curry County). Moreover, since 2003 it has been repeatedly isolated from plants in Oregon nurseries. In this study, we analysed the genetic diversity of the P. ramorum population in Oregon from 2001 to 2004 by using microsatellites. A total of 323 isolates (272 from the infested forest; 51 from nurseries) were screened at 10 loci. The overall P. ramorum population in Oregon is characterized by low genetic diversity and has all the hallmarks of an introduced organism. All isolates within the A2 mating type belonged to the same clonal lineage and no recombinant genotypes were found. The forest population (24 genotypes) was dominated by a single multilocus genotype which persisted over years, indicating that eradication efforts in the forest have not completely eliminated inoculum sources. In contrast, genotypic evidence suggests that eradication was effective in nurseries. In 2003 and 2004, a total of 11 genotypes were found in the nurseries (one belonged to the European lineage of P. ramorum) but no genotype was recovered in both sampling years. Significant differentiation and low gene flow were detected between nursery and forest populations. Only two nursery genotypes were also found in the forest, and then at low frequency. Thus, the nursery infestation is not caused by the genotypes observed in Curry County, but likely resulted through introduction of novel genotypes from nurseries out-of-state. This highlights the continued importance of sanitation and quarantine in nurseries to prevent further introduction and spread of P. ramorum.     

Plant/pathogen interactions observed during host range testing of the rust fungus Uromyces pencanus,a classical biological control agent for Chilean needle grass (Nassella neesiana) in Australia and New Zealand

Nassella neesiana (Chilean needle grass) is a South American grass species that is a serious weed in Australia and New Zealand. The rust fungus Uromyces pencanus is a promising biocontrol agent that could be used to control the weed in both countries. Extensive host range testing has been conducted to explore the specificity of the rust. In this paper we discuss the different degrees of invasion by the rust of the tissues of target and non-target species; the plant defences elicited by such invasion at the cellular level; and their relevance to the biological control of Chilean needle grass.     

Plant microfossil analysis of coprolites of the critically endangered kakapo (Strigops habroptilus) parrot from New Zealand

We present here the results of the analysis of pollen, spores, starch and other microscopic plant material in 52 coprolites of the critically endangered kakapo (Strigops habroptilus), a New Zealand endemic parrot. The six sampling sites (caves, rock overhangs) are in widely separate geographic regions in the South Island, across an altitudinal range encompassing lowland, montane and sub-alpine environments. Radiometric testing gives average ages of discrete coprolite groups ranging from modern to 2514 ± 43 ¹⁴C yr BP (Wk-19164). A wide variety of plant taxa is identified in the coprolites, comprising cones, flowers, leaves and fronds of ~ 30 podocarps, dicotyledonous trees, shrubs and lianas, grasses, ferns, lycopods, mosses and liverworts. This supports previous observations that kakapo are versatile feeders, using a broad spectrum of foods that may only be available for short periods and intermittent years. Many of the food taxa we identify are consistent with the studies of modern kakapo diet. We also identify several new foods, potentially of value in developing food supplements to increase breeding frequency of the current kakapo population. The most commonly identified plant parts in coprolites were fern fronds (monoletes, Hymenophyllum, Cyathea smithii), followed by podocarp cones and leaves. Lack of starch in the coprolites suggests that the lycopod and fern rhizomes reported in modern kakapo diet were not commonly eaten. The data provide initial botanical evidence of diet from coprolites of an extant bird.     

Chemical composition of soil solutions extracted from New Zealand beech forests and West German beech and spruce forests

Concentrations of ions were measured in soil solutions from beech (Nothofagus) forests in remote areas of New Zealand and in solutions from beech (Fagus sylvatica) and Norway spruce (Picea abies) forests in North-East Bavaria, West Germany, to compare the chemistry of soil solutions which are unaffected by acid deposition (New Zealand) with those that are affected (West Germany). In New Zealand, soil solution SO₄ ^2– concentrations ranged between <2 and 58 mol L^–1, and NO₃ ^– concentrations ranged between <1 and 3 mol L^–1. In West Germany, SO₄ ^2– concentrations ranged between 80 and 700 mol L^–1, and NO₃ ^– concentrations at three of six sites ranged between 39 and 3750 mol L^–1, but was not detected at the remaining three sites. At all sites in New Zealand, and at sites where the soil base status was moderately high in West Germany, pH levels increased, and total Al (Al_t) and inorganic monomeric Al (Al_i) levels decreased rapidly with increasing soil depth. In contrast, at sites on soils of low base status in West Germany, pH levels increased only slightly, and Al levels did not decline with increasing soil depth.Under a high-elevation Norway spruce stand showing severe Mg deficiency and dieback symptoms in West Germany, soil solution Mg²⁺ levels ranged between 20 and 60 mol L^–, and were only half those under a healthy stand. Al_t and Al_i levels were substantially higher the healthy stand than under the unhealthy stand, indicating that Al toxicity was not the main cause of spruce decline.     

Population genetic subdivision in the New Zealand greenshell mussel (Perna canaliculus) inferred from single-strand conformation polymorphism analysis of mitochondrial DNA

Single-strand conformation polymorphism (SSCP) analysis of the NADH IV region of the mitochondrial DNA (mtDNA) molecule in greenshell mussels (Perna canaliculus) indicated strong population genetic structuring in this endemic New Zealand species. A northern and a southern group were differentiated by frequency shifts in common haplotypes and by the occurrence of a unique southern haplotype at approximately 20% frequency. This split occurred south of Cook Strait (the body of water between the North and the South Island) at approximately 42 degrees S latitude. Northern populations were less genetically diverse than southern populations and mussels from the west coast of the South Island were most distinct from northern mussels. We hypothesize that the unique haplotype VIII originated in the lower South Island, and that its spread northwards was obstructed by the opening of Cook Strait approximately 15 000-16 000 years ago and the subsequent establishment of present-day surface water circulation patterns in Greater Cook Strait. We suggest that present-day strong tidal flows and turbulent mixing of water masses in Cook Strait, and intense up-welling on the east and west coasts in this region, represent a barrier to gene flow between mussels located in the North Island and northern South Island vs. mussels in most of the South Island and Stewart Island.     

Isozyme analysis of Galaxias species (Teleostei: Galaxiidae) from the Taieri River, South Island, New Zealand: a species complex revealed

We examined genetic differentiation among 23 samples of non-migratory river galaxias from 17 streams in the Taieri River system, South Island, New Zealand. Four major genetic types were found, two of which occur in narrow sympatry in one location. These were compared with topotypical material representing Galaxias anomalus from the Clutha system (Otago) and G. vulgaris from the Waimakariri system (Canterbury) in order to establish identity. Morphological examination of these four major genetic types revealed consistent concomitant differences. The results suggest that there are at least three species of river galaxias in the Taieri system: G. anomalus, G. vulgaris and at least one previously undescribed species. We propose that the genetic structuring and subsequent speciation of this group has been promoted by the absence of the marine juvenile phase that is found in five other members of the genus native to New Zealand. This structuring may be exacerbated by population fragmentation over the last century owing to the negative influence of introduced trout. The phylogenetic diversity within the river system mirrors the diverse flora and invertebrate fauna of the region, and has conservation implications that parallel those resulting from our improved knowledge of the New Zealand herpetofauna through the application of genetic analysis.