Homologous RXLR effectors from Hyaloperonospora arabidopsidis and Phytophthora sojae suppress immunity in distantly related plants

Diverse pathogens secrete effector proteins into plant cells to manipulate host cellular processes. Oomycete pathogens contain large complements of predicted effector genes defined by an RXLR host cell entry motif. The genome of Hyaloperonospora arabidopsidis (Hpa, downy mildew of Arabidopsis) contains at least 134 candidate RXLR effector genes. Only a small subset of these genes is conserved in related oomycetes from the Phytophthora genus. Here, we describe a comparative functional characterization of the Hpa RXLR effector gene HaRxL96 and a homologous gene, PsAvh163, from the Glycine max (soybean) pathogen Phytophthora sojae. HaRxL96 and PsAvh163 are induced during the early stages of infection and carry a functional RXLR motif that is sufficient for protein uptake into plant cells. Both effectors can suppress immune responses in soybean. HaRxL96 suppresses immunity in Nicotiana benthamiana, whereas PsAvh163 induces an HR‐like cell death response in Nicotiana that is dependent on RAR1 and Hsp90.1. Transgenic Arabidopsis plants expressing HaRxL96 or PsAvh163 exhibit elevated susceptibility to virulent and avirulent Hpa, as well as decreased callose deposition in response to non‐pathogenic Pseudomonas syringae. Both effectors interfere with defense marker gene induction, but do not affect salicylic acid biosynthesis. Together, these experiments demonstrate that evolutionarily conserved effectors from different oomycete species can suppress immunity in plant species that are divergent from the source pathogen’s host.     

The genetics of resistance to five races of downy mildew (Plasmopara halstedii) in sunflower (Helianthus annuus L.)

 These studies were undertaken to determine whether downy mildew resistance genes in sunflower were independent as first reported, or linked as suggested by more recent hypotheses. The segregations for downy mildew reaction of 111 F₃ progenies from a cross between a susceptible line and a line with Pl2 were used to locate this gene on the sunflower consensus RFLP linkage map. It was shown that Pl2 was linked to the same RFLP markers on linkage group 1 as Pl1 and Pl6, mapped earlier, and at a very similar distance. The F₃ progenies showed exactly the same segregation patterns when tested with race 1 and race D. One hundred and fifty four progenies from a cross between a susceptible line and HA335, containing Pl6 (considered as giving resistance to all Plasmopara halstedii races), were tested with the five French downy mildew races, 1, A, B, C and D. Two progenies were observed to show segregation for races 1 and D, while appearing homozygous-resistant to races A , B and C. Tests on F₄ progenies confirmed this separation of resistances with fixation of susceptibility to races 1 and D and resistance to races A, B and C. It is concluded that the Pl6 gene is not a “strong” gene, giving resistance to all downy mildew races, but rather a cluster of genes, each providing resistance to one, or a few, downy mildew races. The genes giving resistance to races 1 and D, on one hand, and to races A, B and C, on the other hand, must be very closely linked, with about 0.6 cM between the two groups. Received: 23 December 1996 / Accepted: 18 April 1997     

Interaction between sunflower plants and five isolates of Plasmopara halstedii on the level of pathogenicity

The interaction between sunflower plants showing a high level of quantitative resistance and five Plasmopara halstedii (the causal agent of downy mildew) isolates of several races were studied using five single zoosporangium isolates per pathogen isolate. Aggressiveness criteria were analyzed for 25 P. halstedii single zoosporangium isolates. Based on the reaction for the P. halstedii isolates to four sunflower hybrids H1–H4 varying only in their downy mildew resistance genes, there were differences in virulence spectrum in pathogen isolates. Analysis of five single zoosporangium isolates for P. halstedii isolates showed significant variability within pathogen isolate for all aggressiveness criteria but not for all pathogen isolates. The hypothesis explaining the interaction between P. halstedii and its host plant was discussed on the level of pathogenicity.     

RFLP and RAPD mapping of the sunflower Pl1 locus for resistance to Plasmopara halstedii race 1

The Pl1 locus in sunflower, Helianthus annuus L., conferring resistance to downy mildew, Plasmopara halstedii, race 1 has been located in linkage group 1 of the consensus RFLP map of the cultivated sunflower. Bulked segregant analyses were used on 135 plants of an F₂ progeny from a cross between a downy mildew susceptible line, GH, and RHA266, a line carrying Pl1. Two RFLP markers and one RAPD marker linked to the Pl1 locus have been identified. The RFLP markers are located at 5.6 cM and 7.1 cM on either side of Pl1. The RAPD marker is situated at 43.7 cM from Pl1. The significance and applications of these markers in sunflower breeding are discussed.     

Development of PCR markers for the<Emphasis Type="Italic"> Pl5/Pl8</Emphasis> locus for resistance to<Emphasis Type="Italic"> Plasmopara halstedii</Emphasis> in sunflower,<Emphasis Type="Italic"> Helianthus annuus</Emphasis> L. from complete CC-NBS-LRR sequences

Sunflower downy mildew, caused by Plasmopara halstedii, is one of the major diseases of this crop. Development of elite sunflower lines resistant to different races of this oomycete seems to be the most efficient method to limit downy mildew damage. At least two different gene clusters conferring resistance to different races of P. halstedii have been described. In this work we report the cloning and mapping of two full-length resistance gene analogs (RGA) belonging to the CC-NBC-LRR class of plant resistance genes. The two sequences were then used to develop 14 sequence tagged sites (STS) within the Pl5/Pl8 locus conferring resistance to a wide range of P. halstedii races. These STSs will be useful in marker-assisted selection programs.Communicated by C. Möllers     

Molecular analysis of a major locus for resistance to downy mildew in sunflower with specific PCR-based markers

Resistance of sunflower to the obligate parasite Plasmopara halstedii is conferred by specific dominant genes, denoted Pl. The Pl6 locus confers resistance to all races of P. halstedii except one, and must contain at least 11 tightly linked genes each giving resistance to different downy mildew races. Specific primers were designed and used to amplify 13 markers covering a genetic distance of about 3 cM centred on the Pl6 locus. Cloning and sequence analysis of these 13 markers indicate that Pl6 contains conserved genes belonging to the TIR-NBS-LRR class of plant resistance genes. Received: 9 April 2001 / Accepted: 10 August 2001     

An RXLR effector PlAvh142 from Peronophythora litchii triggers plant cell death and contributes to virulence

Litchi downy blight, caused by the phytopathogenic oomycete Peronophythora litchii, results in tremendous economic loss in litchi production every year. To successfully colonize the host cell, Phytophthora species secret hundreds of RXLR effectors that interfere with plant immunity and facilitate the infection process. Previous work has already predicted 245 candidate RXLR effector-encoding genes in P. litchii, 212 of which have been cloned and tested for plant cell death-inducing activity in this study. We found three such RXLR effectors could trigger plant cell death through transient expression in Nicotiana benthamiana. Further experiments demonstrated that PlAvh142 could induce cell death and immune responses in several plants. We also found that PlAvh142 localized in both the cytoplasm and nucleus of plant cells. The cytoplasmic localization was critical for its cell death-inducing activity. Moreover, deletion either of the two internal repeats in PlAvh142 abolished the cell death-inducing activity. Virus-induced gene silencing assays showed that cell death triggered by PlAvh142 was dependent on the plant transduction components RAR1 (require for Mla12 resistance), SGT1 (suppressor of the G2 allele of skp1) and HSP90 (heat shock protein 90). Finally, knockout of PlAvh142 resulted in significantly attenuated P. litchii virulence on litchi plants, whereas the PlAvh142-overexpressed mutants were more aggressive. These data indicated that PlAvh142 could be recognized in plant cytoplasm and is an important virulence RXLR effector of P. litchii.     

Antisense Expression of a NBS-LRR Sequence in Sunflower (Helianthus annuus L.) and Tobacco (Nicotiana tabacum L.): Evidence for a Dual Role in Plant Development and Fungal Resistance

A partial sunflower cDNA clone, PLFOR48, segregating with a resistance marker to Plasmopara halstedii, the causal agent of downy mildew, has been cloned from the mildew resistant sunflower line, RHA 266. PLFOR48 encodes a putative protein with a nucleotide-binding site and a leucine-rich repeat domain, showing significant homology with previously cloned resistance genes belonging to the TIR-NBS-LRR family. Southern blot analysis of non-transgenic sunflower suggests that PLFOR48 is part of a multigenic family. The potential role of PLFOR48 sequence in sunflower resistance to mildew was studied, by assessing loss of function, using expression of the antisense cDNA in RHA 266 sunflower line. Quite unexpectedly, transgenic sunflower lines displayed severe developmental abnormalities, and in particular, on the main meristems of homozygote T2 progeny, thus hampering any further challenge inoculation with Plasmopara halstedii. The presence of homologous sequences to PLFOR48 in Nicotiana tabacum var Samsun NN, as demonstrated by Southern blotting, drove us to consider tobacco as an additional model to investigate the potential role of this sequence in fungal resistance. Expression of the same antisense cDNA in transgenic tobacco lines gave rise to higher degree of susceptibility to Phytophthora parasitica, as well as to severe alterations in seed development. These results suggest that PLFOR48 and homologous sequences could be involved in both regulating developmental pathways and controlling resistance to fungal pathogens.     

An RXLR effector secreted by Phytophthora parasitica is a virulence factor and triggers cell death in various plants

RXLR effectors encoded by Phytophthora species play a central role in pathogen–plant interactions. An understanding of the biological functions of RXLR effectors is conducive to the illumination of the pathogenic mechanisms and the development of disease control strategies. However, the virulence function of Phytophthora parasitica RXLR effectors is poorly understood. Here, we describe the identification of a P. parasitica RXLR effector gene, PPTG00121 (PpE4), which is highly transcribed during the early stages of infection. Live cell imaging of P. parasitica transformants expressing a full-length PpE4 (E4FL)-mCherry protein indicated that PpE4 is secreted and accumulates around haustoria during plant infection. Silencing of PpE4 in P. parasitica resulted in significantly reduced virulence on Nicotiana benthamiana. Transient expression of PpE4 in N. benthamiana in turn restored the pathogenicity of the PpE4-silenced lines. Furthermore, the expression of PpE4 in both N. benthamiana and Arabidopsis thaliana consistently enhanced plant susceptibility to P. parasitica. These results indicate that PpE4 contributes to pathogen infection. Finally, heterologous expression experiments showed that PpE4 triggers non-specific cell death in a variety of plants, including tobacco, tomato, potato and A. thaliana. Virus-induced gene silencing assays revealed that PpE4-induced cell death is dependent on HSP90, NPK and SGT1, suggesting that PpE4 is recognized by the plant immune system. In conclusion, PpE4 is an important virulence RXLR effector of P. parasitica and recognized by a wide range of host plants.     

Different pathotypes of the sunflower downy mildew pathogen Plasmopara halstedii all contain isometric virions†

Eight pathotypes of Plasmopara halstedii were screened to investigate the occurrence of virions and the potential viral influence on the pathogenicity of the sunflower downy mildew pathogen. In 23 of 26 P. halstedii isolates derived from eight countries in Europe, North America and South America, virions were detected by transmission electron microscopy. By contrast, there were no ultrastructural indications of virus‐like particles in eight other related Oomycetes. The virions of representative P. halstedii isolates were morphologically and biochemically characterized and compared among each other. Regardless of their host's pathotypes, the geographical origin of the isolate and the sensitivity towards the fungicide metalaxyl, the viral characters obtained were uniform. The virions were isometric and measured approximately 37 nm in diameter. One polypeptide of c. 36 kDa and two segments of single‐stranded RNA (3.0 and 1.6 kb) were detected. Both viral RNA segments were detected by capillary electrophoresis in the three remaining P. halstedii isolates where virions were undetectable by transmission electron microscopy. Virus‐specific primers for the 1.6 kb‐segment were synthesized and used to determine and compare a partial sequence of the viral coat protein among virions of different P. halstedii pathotypes. In all tested isolates, fragments of 0.7 kb were amplified which were directly sequenced. Sequence variation was insignificant. As both less aggressive and more aggressive P. halstedii isolates contained virions, the presence or absence of virions could not explain the diverse aggressiveness of the downy mildew pathogen towards sunflower. Moreover, the results indicated that pathogenicity of P. halstedii was not related to variation in morphological or biochemical characters of the virions.     

Identification of Avramr1 from Phytophthora infestans using long read and cDNA pathogen-enrichment sequencing (PenSeq)

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, significantly hampers potato production. Recently, a new Resistance to Phytophthora infestans (Rpi) gene, Rpi-amr1, was cloned from a wild Solanum species, Solanum americanum. Identification of the corresponding recognized effector (Avirulence or Avr) genes from P. infestans is key to elucidating their naturally occurring sequence variation, which in turn informs the potential durability of the cognate late blight resistance. To identify the P. infestans effector recognized by Rpi-amr1, we screened available RXLR effector libraries and used long read and cDNA pathogen-enrichment sequencing (PenSeq) on four P. infestans isolates to explore the untested effectors. Using single-molecule real-time sequencing (SMRT) and cDNA PenSeq, we identified 47 highly expressed effectors from P. infestans, including PITG_07569, which triggers a highly specific cell death response when transiently coexpressed with Rpi-amr1 in Nicotiana benthamiana, suggesting that PITG_07569 is Avramr1. Here we demonstrate that long read and cDNA PenSeq enables the identification of full-length RXLR effector families and their expression profile. This study has revealed key insights into the evolution and polymorphism of a complex RXLR effector family that is associated with the recognition by Rpi-amr1.     

Dissecting virulence function from recognition: cell death suppression in Nicotiana benthamiana by XopQ/HopQ1‐family effectors relies on EDS1‐dependent immunity

Many Gram‐negative plant pathogenic bacteria express effector proteins of the XopQ/HopQ1 family which are translocated into plant cells via the type III secretion system during infection. In Nicotiana benthamiana, recognition of XopQ/HopQ1 proteins induces an effector‐triggered immunity (ETI) reaction which is not associated with strong cell death but renders plants immune against Pseudomonas syringae and Xanthomonas campestris pv. vesicatoria strains. Additionally, XopQ suppresses cell death in N. benthamiana when transiently co‐expressed with cell death inducers. Here, we show that representative XopQ/HopQ1 proteins are recognized similarly, likely by a single resistance protein of the TIR‐NB‐LRR class. Extensive analysis of XopQ derivatives indicates the recognition of structural features. We performed Agrobacterium‐mediated protein expression experiments in wild‐type and EDS1‐deficient (eds1) N. benthamiana leaves, not recognizing XopQ/HopQ1. XopQ recognition limits multiplication of Agrobacterium and attenuates levels of transiently expressed proteins. Remarkably, XopQ fails to suppress cell death reactions induced by different effectors in eds1 plants. We conclude that XopQ‐mediated cell death suppression in N. benthamiana is due to the attenuation of Agrobacterium‐mediated protein expression rather than the cause of the genuine XopQ virulence activity. Thus, our study expands our understanding of XopQ recognition and function, and also challenges the commonly used co‐expression assays for elucidation of in planta effector activities, at least under conditions of ETI induction.     

Recognition of lettuce downy mildew effector BLR38 in Lactuca serriola LS102 requires two unlinked loci

Plant-pathogenic oomycetes secrete effector proteins to suppress host immune responses. Resistance proteins may recognize effectors and activate immunity, which is often associated with a hypersensitive response (HR). Transient expression of effectors in plant germplasm and screening for HR has proven to be a powerful tool in the identification of new resistance genes. In this study, 14 effectors from the lettuce downy mildew Bremia lactucae race Bl:24 were screened for HR induction in over 150 lettuce accessions. Three effectors—BLN06, BLR38 and BLR40—were recognized in specific lettuce lines. The recognition of effector BLR38 in Lactuca serriola LS102 did not co-segregate with resistance against race Bl:24, but was linked to resistance against multiple other B. lactucae races. Two unlinked loci are both required for effector recognition and are located near known major resistance clusters. Gene dosage affects the intensity of the BLR38-triggered HR, but is of minor importance for disease resistance.     

Relationship between virulence and aggressiveness in Plasmopara halstedii (sunflower downy mildew)

Relationship between virulence and aggressiveness was studied in seven Plasmopara halstedii (sunflower downy mildew) pathotypes including five progeny pathotypes of races 300, 304, 314, 704 and 714 arising from two parental pathotypes of races 100 and 710. Aggressiveness criteria including percentage infection, latent period, sporulation density and reduction of hypocotyl length were analysed in one sunflower inbred line showing a high level of quantitative resistance. There were significant differences between P. halstedii pathotypes for all aggressiveness criteria. Pathogenicity of progeny pathotypes as compared with parental ones (relationship between virulence and aggressiveness) seems to be positive, negative or uncorrelated. Hypothesis explaining these cases are discussed.     

Induction of resistance against downy mildew on sunflower by rhizobacteria

Abstract

Induction of resistance to downy mildew caused by Plasmopara halstedii in sunflower was studied after treatment with PGPR (plant growth promoting rhizobacteria) strain INR7 (Bacillus spp). Treatment of sunflower seeds with 1×10⁸cfu/ml of PGPR strain INR7 resulted in decreased disease severity and offered 51 and 54% protection under green house and field conditions, respectively. The induction of resistance to P. halstedii by PGPR strain INR7 was accompanied by the accumulation of various host defense-related enzymes in susceptible sunflower seedlings. Enhanced activation of catalase (CAT), phenylalanine ammonia-lyase (PAL), peroxidase (POX), polyphenol oxidase (PPO) and chitinase (CHI) was evident at 6, 9, 12, 12 and 12h post inoculation, respectively, in sunflower seedlings raised from seeds treated with PGPR strain INR7. This enhanced and early activation of defense-related responses in the susceptible cultivar after treatment with PGPR strain INR7 was comparable to that in the resistant cultivar. The results indicate that PGPR strain INR7 induced resistance against P. halstedii in sunflower is mediated through enhanced expression of defense mechanism.     

Cloning of molecular markers for disease resistance in sunflower, Helianthus annuus L.

 A candidate-gene approach to analyse the resistance of plants to phytopathogenic fungi is presented. The resistance of sunflower (Helianthus annuus L.) to downy mildew (Plasmopara halstedii) shows a gene-for-gene interaction (monogenic resistance), whereas resistance to white rot (Sclerotinia sclerotiorum) is quantitative, with different levels of resistance for different plant parts. By homology cloning, probes were obtained homologous to some plant resistance genes (nucleotide binding site-like, NBS, genes and serine-threonine protein kinase-like, PK, genes). These clones were used as probes for linkage mapping of the corresponding genes. It was demonstrated that at least three NBS-like loci are located on linkage-group 1, in the region where downy mildew resistance loci have been described. Quantitative trait loci for S. sclerotiorum resistance to penetration or extension of the mycelium in different tissues were studied in three crosses. Major QTLs for resistance were found on linkage group 1, with up to 50% of the phenotypic variability explained by peaks at the map position of the PK locus, 25 cM from the downy mildew loci. Received: 24 September 1997 / Accepted: 21 October 1997     

Twelve polymorphic expressed sequence tags‐derived markers for Plasmopara halstedii,the causal agent of sunflower downy mildew

Twelve expressed sequence tags‐derived markers were isolated from Plasmopara halstedii (Oomycetes), the causal agent of sunflower downy mildew. A total of 25 single nucleotide polymorphisms and five indels were detected by single‐strand conformation polymorphism analysis and developed for high‐throughput genotyping of 32 isolates. There was a high level of genetic diversity (H_E = 0.484). Observed heterozygosity ranged from 0 to 0.143 indicating that P. halstedii is probably a selfing species. These markers were also useful in detecting significant genetic variations among French populations (F_ST = 0.193) and between French and Russian populations (F_ST = 0.23). Cross‐amplification tests on three closely related species indicated that no loci amplified in other Oomycete species.