‘Plantibodies’: a flexible approach to design resistance against pathogens

Engineering resistance against various diseases and pests is hampered by the lack of suitable genes. To overcome this problem we started a research program aimed at obtaining resistance by transfecting plants with genes encoding monoclonal antibodies against pathogen specific proteins. The idea is that monoclonal antibodies will inhibit the biological activity of molecules that are essential for the pathogenesis. Potato cyst nematodes are chosen as a model and it is thought that monoclonal antibodies are able to block the function of the saliva proteins of this parasite. These proteins are, among others, responsible for the induction of multinucleate transfer cells upon which the nematode feeds. It is well documented that the ability of antibodies to bind molecules is sufficient to inactivate the function of an antigen and in view of the potential of animals to synthesize antibodies to almost any molecular structure, this strategy should be feasible for a wide range of diseases and pests.Antibodies have several desirable features with regard to protein engineering. The antibody (IgG) is a Y-shaped molecule, in which the domains forming the tips of the arms bind to antigen and those forming the stem are responsible for triggering effector functions (Fc fragments) that eliminate the antigen from the animal. Domains carrying the antigen-binding loops (Fv and Fab fragments) can be used separately from the Fc fragments without loss of affinity. The antigen-binding domains can also be endowed with new properties by fusing them to toxins or enzymes. Antibody engineering is also facilitated by the Polymerase Chain Reaction (PCR). A systematic comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the 3-ends, but also the 5-ends of the antibody genes are relatively conserved. We were able to design a small set of primers with restriction sites for forced cloning, which allowed the amplification of genes encoding antibodies specific for the saliva proteins ofGlobodera rostochiensis. Complete heavy and light chain genes as well as single chain Fv fragments (scFv), in which the variable parts of the light (V_L) and heavy chain (V_H) are linked by a peptide, will be transferred to potato plants. A major challenge will be to establish a correct expression of the antibody genes with regard to three dimensional folding, assembly and intracellular location.     

Priming: it’s all the world to induced disease resistance

After infection by a necrotising pathogen, colonisation of the roots with certain beneficial microbes, or after treatment with various chemicals, many plants establish a unique physiological situation that is called the ‘primed’ state of the plant. In the primed condition, plants are able to ‘recall’ the previous infection, root colonisation or chemical treatment. As a consequence, primed plants respond more rapidly and/or effectively when re-exposed to biotic or abiotic stress, a feature that is frequently associated with enhanced disease resistance. Though priming has been known as a component of induced resistance for a long time, most progress in the understanding of the phenomenon has been made over the past few years. Here we summarize the current knowledge of priming and its relevance for plant protection in the field.     

Detection of the Phoma pathogens Plenodomus biglobosus subclades ‘brassicae’ and ‘canadensis’ on wasabi,and ‘canadensis’ in Europe

European Journal of Plant Pathology - Phoma stem canker / blackleg is an internationally important disease of Brassicas including B. napus (oilseed rape, OSR), caused by multiple genetic subclades...     

Comment on ‘In Turkish wheat cultivars the resistance allele of LR34 is ineffective against leaf rust’

Journal of Plant Diseases and Protection -     

Lysobacter gummosus OH17 induces systemic resistance in Oryza sativa ‘Nipponbare’

Although rice is one of the most cultivated, consumed, and essential crops worldwide, it is highly susceptible to a wide range of bacterial and fungal pathogens that significantly reduce the production and quality of rice. Recently, our research group reported that the plant growth-promoting rhizobacterium Lysobacter gummosus OH17 was able to enhance the ethylene levels in Oryza sativa ‘Nipponbare’ plants at the late interaction stages. In this work, L. gummosus OH17 was found to be capable of inducing the overexpression of relevant genes of the jasmonic acid and ethylene transduction pathways in Nipponbare plants, such as OsACC, OsACO, OsERF3, and OsLOX, which resulted in the up-regulation of a number of pathogenesis-related proteins. The observed metabolic effects enhanced the disease resistance of rice against the three most devastating rice pathogens: Magnaporthe oryzae causing rice blast, Rhizoctonia solani causing rice sheath blight, and Xanthomonas oryzae pv. oryzae causing bacterial leaf blight. Furthermore, it was shown that L. gummosus OH17 also enhanced ethylene production levels in other O. sativa varieties from both the japonica and indica subspecies. Here, we report for the first time the metabolic alterations produced by plant growth-promoting rhizobacterium L. gummosus OH17 at the late interaction stages and how these alterations induce systemic resistance.     

Hexanoic acid provides long-lasting protection in ‘Fortune’ mandarin against Alternaria alternata

Alternaria brown spot disease is a serious disease in mandarins and their hybrids without effective disease control measures. In recent years, induced plant resistance has been studied as an alternative to classical pesticides, but few studies on the effectiveness of these products and their long-lasting effects in woody crops have been performed. After two inoculations with Alternaria alternata, citrus plants that were treated with hexanoic acid showed enhanced resistance, displaying lower levels of disease incidence associated with an activation of the jasmonic acid pathway, the accumulation of phenolic compounds and the expression of defensive genes, such as polygalacturonase-inhibiting proteins.     

African wheat germplasm – a valuable resource for resistance to rust diseases

Known and unknown genes conferring seedling and adult plant resistance (APR) to leaf rust, stem rust and stripe rust were detected either singly or in combination in a set of 136 African wheat genotypes using multi-pathotype tests with characterized Australian Puccinia triticina (Pt), P. graminis f. sp. tritici (Pgt) and P. striiformis f. sp. tritici (Pst) pathotypes. Lines Beladi 132, IYN 68/9.44, Kenya Kifaru and Kenya Mbweha were postulated to carry resistance against multiple pathotypes of Pt, Pgt and Pst, whereas IAR/W/163-3, Grano Di Moggio Tipo 44 and Trigo 48 had resistance against all pathotypes tested in the current study. Field evaluation with the three rust pathogens detected low to high APR in more than 50% of lines, and while most tested positive with markers linked to known APR genes (csLV34, csLV46G22, TM10KASPAR, csGS, Cfb5006 and csSr2), many carried unidentified and useful resistance to all three rusts. Genetic analysis of F₃ mapping populations based on seven genotypes showed either monogenic or digenic inheritance of APR to leaf rust, stem rust and stripe rust. The lines postulated to carry effective uncharacterized seedling genes and APR genes are of great potential value in diversifying resistance to help achieve durable control of all three rust diseases of wheat.     

Identifying potential novel resistance to the foliar disease ‘Scald’ (Rhynchosporium commune) in a population of Scottish Bere barley landrace (Hordeum vulgare L.)

Journal of Plant Diseases and Protection - Barley ‘Scald’ is an economically damaging fungal disease that is a global problem, causing significant yield and economical losses in the UK...     

Seasonal colonisation of apple trees by ‘Candidatus Phytoplasma mali’ revealed by a new quantitative TaqMan real-time PCR approach

Apple proliferation (AP), caused by ‘Candidatus Phytoplasma mali’, is an economically important disease affecting many apple-growing areas in Europe. A new TaqMan real-time PCR assay was established for absolute quantification of ‘Ca. P. mali’ by using a single-copy gene of the host plant as a reference, which is amplified with the pathogen DNA in a single-tube reaction. Normalised estimates of phytoplasma concentration are ultimately expressed as the number of phytoplasma cells per host plant cell. The assay was used to monitor the ‘Ca. P. mali’ titre over the course of two growing seasons in roots and branches of symptomatic and asymptomatic but AP-positive apple trees. All 252 root samples from symptomatic and asymptomatic trees tested positive, with an average number of 59.8 ± 5.68 (standard error) and 55.1 ± 9.83 ‘Ca. P. mali’ per host cell, respectively. From the 378 shoot samples analysed, 81% of the symptomatic and only 20% of the asymptomatic samples were AP-positive with an average number of 9.4 ± 1.04 and 0.7 ± 0.13 ‘Ca. P. mali’ per host cell, respectively. This strengthens evidence that not the pathogen occurrence alone but the presence of a certain quantity of ‘Ca. P. mali’ in the aerial tree sections is involved in symptom expression. In addition, pronounced seasonality of the phytoplasma concentration was found, not only in branches, but also for the first time in roots of symptomatic and asymptomatic apple trees. Highest phytoplasma levels in roots were detected from December to May.     

Identification of Brassica accessions resistant to ‘old’ and ‘new’ pathotypes of Plasmodiophora brassicae from Canada

Genetic resistance is the main tool used to manage clubroot of canola (Brassica napus) in Canada. However, the emergence of new virulent strains of the clubroot pathogen, Plasmodiophora brassicae, has complicated canola breeding efforts. In this study, 386 Brassica accessions were screened against five single-spore isolates (represented by pathotypes 2F, 3H, 5I, 6M and 8N on the Canadian Clubroot Differential Set) and 17 field isolates (represented by 12 unique pathotypes: 2B, 3A, 3D, 3O, 5C, 5G, 5K, 5L, 5X, 8E, 8J and 8P) of P. brassicae to identify resistance sources effective against these strains. The results showed that one B. rapa accession (CDCNFG-046, mean index of disease (ID) = 3.3%) and two B. nigra accessions (CDCNFG-263, mean ID = 3.1%; and CDCNFG-262, mean ID = 4.7%) possessed excellent resistance to all 22 of the isolates evaluated. Fifty other accessions showed differential clubroot reactions (resistant, moderately resistant or susceptible), including 27 (one B. napus, two B. rapa, four B. oleracea and 20 B. nigra) accessions that were each resistant to 8–21 P. brassicae isolates, but developed mean IDs in the range of 5.3–29.6%. The remaining 23 accessions (two B. napus, one B. rapa, five B. oleracea and 15 B. nigra) were each resistant to 3–13 isolates, but developed mean IDs in the range of 30.3–47.0%. The three accessions that showed absolute resistance and the 50 accessions that showed differential clubroot reactions could be used to breed for resistance to the new P. brassicae strains.     

Comparative resistance to foliar fungal pathogens in transgenic carrot plants expressing genes encoding for chitinase, β-1,3-glucanase and peroxidise

Genes encoding an acidic wheat class IV chitinase (383), an acidic wheat β 1,3-glucanase (638) and a rice cationic peroxidase (POC1) were introduced into ‘Nantes Coreless’ carrot (Daucus carota) by Agrobacterium-mediated transformation. The genes were introduced singly or in various combinations followed by selection imposed by the herbicide phosphinothricin. Regenerated plantlets were screened for presence and expression of the three transgenes using PCR, Southern and Northern hybridisations. Eighteen transgenic lines expressing a single transgene and 2 lines each co-expressing 638/383 and 383/POC1 were assessed for resistance to the necrotrophic fungal pathogens Botrytis cinerea and Sclerotinia sclerotiorum. Percentage leaf area diseased was measured 4 and 7 days after inoculation (dai) and compared to non-transformed control plants. Six lines expressing β-1,3-glucanase 638 alone had no enhanced resistance to B. cinerea at 4 dai and only slight resistance to S. sclerotiorum; there was no effect at 7 dai. Two out of the six lines expressing 383 alone had enhanced tolerance to both pathogens with a 20–50% reduction in disease development at 7 dai. Two lines co-expressing 638/383 had slight reductions in disease by (10–20%) similar to that of the lines expressing chitinase 383 alone. Highest levels of disease resistance were seen in transgenic lines expressing POC1, alone or in combination with chitinase 383. Disease symptoms were slower to develop and symptoms were reduced by up to 90% for B. cinerea and 70% for S. sclerotiorum. The 383/POC1 co-expressing plants developed disease at levels similar to that of POC1 alone. Petioles of plants over-expressing POC1 had higher levels of lignin accumulation constitutively compared to control plants, which was greatly enhanced following inoculation with S. sclerotiorum. These results indicate that peroxidase over-expression can lead to significant disease reduction against necrotrophic pathogens in transgenic carrot plants.     

Correction to: Haplotypes of ‘Candidatus Liberibacter solanacearum’ identified in Umbeliferous crops in Spain

European Journal of Plant Pathology -     

Spider mite control and resistance management: does a genome help?

The complete genome of the two‐spotted spider mite, Tetranychus urticae, has been reported. This is the first sequenced genome of a highly polyphagous and resistant agricultural pest. The question as to what the genome offers the community working on spider mite control is addressed. Copyright © 2012 Society of Chemical Industry     

β-Tubulins in Gibberella zeae: their characterization and contribution to carbendazim resistance

BACKGROUND: Fusarium head blight caused by Gibberella zeae is an important disease of wheat and barley because it reduces grain yield and quality and results in the contamination of grain with mycotoxins. Recent studies have shown that carbendazim resistance in field strains of G. zeae is not caused by mutation of the β‐tubulin gene (β₁tub), which is the case with other filamentous fungi, but that fungicide resistance is greatly increased by deletion of β₁tub. The aim of the present study was to clarify the function of β₁tub and its role in carbendazim resistance in G. zeae by artificial gene operation. RESULTS: Deletion of β₁tub reduced vegetative growth and pathogenicity but increased asexual reproduction in G. zeae. All the mutants were more resistant to carbendazim than parent strains. A three‐dimensional model of β₁tub was constructed, and the possible carbendazim binding site was analysed. CONCLUSION: β₁tub is not an essential gene in G. zeae, but it affects the sensitivity of the fungus to carbendazim. Copyright © 2012 Society of Chemical Industry     

Plant and veterinary disease diagnosis: a generic approach to the development of field tools for rapid decision making?

Rapid and accurate diagnostic tests make an important contribution to programmes to monitor and eradicate infectious diseases that impact animal and plant health. Using foot-and-mouth disease (FMD) and sudden oak death as examples, this review outlines recent progress to develop new field tools for detection of the infectious agents that cause high-impact livestock and plant diseases. The principal driver for this work is to develop tools that can be used locally to assist in decision making. Advances in this area have developed simple-to-use lateral-flow devices for the detection of FMD virus and the genus Phytophthora (including Phytophthora ramorum , the causal agent of sudden oak death and the related pathogen P. kernoviae ), as well as new hardware platforms to allow PCR testing for these agents by non-specialists in the field. Although developed for different diseases, the user requirements for rapid diagnostic tools for FMD and sudden oak death share many similarities. Using generic solutions to these challenging problems, it is now possible to imagine a new paradigm for how the collection and testing of samples to monitor the spread of important livestock and plant diseases might be achieved.     

Bt-transgenic crops: just another pretty insecticide or a chance for a new start in resistance management?

Transgenic insect-resistant crops carrying genes from Bacillus thuringiensis were grown commercially for the first time in 1996 amid considerable public controversy about resistance management. Several resistance management strategies have been proposed for Bt-transgenic crops. The most promising with currently available technology is the use of refuges of non-transgenic crops, augmented where possible with high toxin expression in the plant and avoiding mosaics of different toxins and pesticides. One problem is that the refuge sizes that are seen as commercially and practically acceptable are generally too small to provide a comfortable margin for the delay of resistance. A promising long-term strategy for delaying resistance, and one which is more forgiving on refuge size, is the pyramiding of two or more insecticidal genes in the same plant. The critical limiting factor to resistance management for transgenic crops will be implementation, which will require cooperation among companies. ©1997 SCI     

Anticoagulant resistance: a relevant issue in sewer rat (Rattus norvegicus) control?

BACKGROUND: The majority of rat problems in cities are thought to be related to defective sewers, and the use of anticoagulant rodenticides in such places is often implemented as part of regular urban rodent control. Knowledge pertaining to the resistance status of sewer rat populations is non‐existent, which may be leading to control problems in cities. It has become crucial to provide knowledge on the prevalence of resistance and how different control strategies have affected its prevalence among sewer rat populations. The prevalence of resistance was investigated in six sewer locations in Copenhagen and its suburban area by means of the blood clotting response (BCR) test and amplification refractory mutation system polymerase chain reaction (ARMS PCR) analysis, and by additional sequencing of the VKORC1 gene. The sewer locations were chosen to represent three different control strategies: (i) no anticoagulant use for approximately 20 years; (ii) no anticoagulant use for the last 5 years; (iii) continuous use for several decades up to the present. RESULTS: A low level of anticoagulant resistance was found in the sewers regardless of control strategy. Surprisingly, none of the rats, including the resistant rats, had resistance‐related mutations in the VKORC1 gene. CONCLUSION: The results of this study suggest that the genetic background of anticoagulant resistance may have to be redefined in respect of resistance‐related changes in the VKORC1 gene. Copyright © 2009 Society of Chemical Industry