A potato late blight resistance gene protects against multiple Phytophthora species by recognizing a broadly conserved RXLR-WY effector.

Species of the genus Phytophthora, the plant killer, cause disease and reduce yields in many crop plants. Although many Resistance to Phytophthora infestans (Rpi) genes effective against potato late blight have been cloned, few have been cloned against other Phytophthora species. Most Rpi genes encode nucleotide-binding domain, leucine-rich repeat-containing (NLR) immune receptor proteins that recognize RXLR (Arg-X-Leu-Arg) effectors. However, whether NLR proteins can recognize RXLR effectors from multiple Phytophthora species has rarely been investigated. Here, we identified a new RXLR-WY effector AVRamr3 from P. infestans that is recognized by Rpi-amr3 from a wild Solanaceae species Solanum americanum. Rpi-amr3 associates with AVRamr3 in planta. AVRamr3 is broadly conserved in many different Phytophthora species, and the recognition of AVRamr3 homologs by Rpi-amr3 activates resistance against multiple Phytophthora pathogens, including the tobacco black shank disease and cacao black pod disease pathogens P. parasitica and P. palmivora. Rpi-amr3 is thus the first characterized resistance gene that acts against P. parasitica or P. palmivora. These findings suggest a novel path to redeploy known R genes against different important plant pathogens.

Natural Genetic Diversity in the Potato Resistance Gene RB Confers Suppression Avoidance from Phytophthora Effector IPI-O4.

RB is a potato gene that provides resistance to a broad spectrum of genotypes of the late blight pathogen Phytophthora infestans. RB belongs to the CC-NB-LRR (coiled-coil, nucleotide-binding, leucine-rich repeat) class of resistance (R) genes, a major component of the plant immune system. The RB protein detects the presence of class I and II IPI-O effectors from P. infestans to initiate a hypersensitive resistance response, but this activity is suppressed in the presence of the Class III effector IPI-O4. Using natural genetic variation of RB within potato wild relatives, we identified two amino acids in the CC domain that alter interactions needed for suppression of resistance by IPI-O4. We have found that separate modification of these amino acids in RB can diminish or expand the resistance capability of this protein against P. infestans in both Nicotiana benthamiana and potato. Our results demonstrate that increased knowledge of the molecular mechanisms that determine resistance activation and R protein suppression by effectors can be utilized to tailor-engineer genes with the potential to provide increased durability.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Phytophthora infestans (Late blight) Infection Assay in a Detached Leaf of Potato.

Phytophthora infestans is a hemibiotroph oomycete that primarily infects potato and tomato. It infects stems, leaves, and tubers and fruits of potato and tomato. High throughput and reproducible infection assays are prerequisites to find sources of resistance in any crop. In this protocol, we describe a detached leaf assay (DLA) for conducting the virulence assay of P. infestans in potato leaves. A late blight infection assay using a potato detached leaf is a semi-high throughput assay in which hundreds of plants can be screened in a laboratory setting.

A complex resistance locus in Solanum americanum recognizes a conserved Phytophthora effector.

Late blight caused by Phytophthora infestans greatly constrains potato production. Many Resistance (R) genes were cloned from wild Solanum species and/or introduced into potato cultivars by breeding. However, individual R genes have been overcome by P. infestans evolution; durable resistance remains elusive. We positionally cloned a new R gene, Rpi-amr1, from Solanum americanum, that encodes an NRC helper-dependent CC-NLR protein. Rpi-amr1 confers resistance in potato to all 19 P. infestans isolates tested. Using association genomics and long-read RenSeq, we defined eight additional Rpi-amr1 alleles from different S. americanum and related species. Despite only ~90% identity between Rpi-amr1 proteins, all confer late blight resistance but differentially recognize Avramr1 orthologues and paralogues. We propose that Rpi-amr1 gene family diversity assists detection of diverse paralogues and alleles of the recognized effector, facilitating durable resistance against P. infestans.

Screening of Wild Potatoes Identifies New Sources of Late Blight Resistance.

Late blight (LB) of potato is considered one of the most devastating plant diseases in the world. Most cultivated potatoes are susceptible to this disease. However, wild relatives of potatoes are an excellent source of LB resistance. We screened 384 accessions of 72 different wild potato species available from the U.S. Potato GeneBank against the LB pathogen Phytophthora infestans in a detached leaf assay (DLA). P. infestans isolates US-23 and NL13316 were used in the DLA to screen the accessions. Although all plants in 273 accessions were susceptible, all screened plants in 39 accessions were resistant. Resistant and susceptible plants were found in 33 accessions. All tested plants showed a partial resistance phenotype in two accessions, segregation of resistant and partial resistant plants in nine accessions, segregation of partially resistant and susceptible plants in four accessions, and segregation of resistant, partially resistant, and susceptible individuals in 24 accessions. We found several species that were never before reported to be resistant to LB: Solanum albornozii, S. agrimoniifolium, S. chomatophilum, S. ehrenbergii, S. hypacrarthrum, S. iopetalum, S. palustre, S. piurae, S. morelliforme, S. neocardenasii, S. trifidum, and S. stipuloideum. These new species could provide novel sources of LB resistance. P. infestans clonal lineage-specific screening of selected species was conducted to identify the presence of RB resistance. We found LB resistant accessions in Solanum verrucosum, Solanum stoloniferum, and S. morelliforme that were susceptible to the RB overcoming isolate NL13316, indicating the presence of RB-like resistance in these species.

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.

Identification of new regulatory genes involved in the pathogenic functions of the rice-pathogenic bacterium Burkholderia glumae.

Burkholderia glumae is an emerging plant-pathogenic bacterium that causes disease in rice in several of the major rice-producing areas throughout the world. In the southern United States, B. glumae is the major causal agent of bacterial panicle blight of rice and has caused severe yield losses in recent decades. Despite its importance, few management options are available for diseases caused by B. glumae, and knowledge of how this pathogen causes disease is limited. In an effort to identify novel factors that contribute to the pathogenicity of B. glumae, random mutagenesis using the miniTn5gus transposon was performed on two strains of B. glumae. Resultant mutants were screened in the laboratory for altered phenotypes in various known or putative virulence factors, including toxoflavin, lipase and extracellular polysaccharides. Mutants that exhibited altered phenotypes compared to their parent strain were selected and subsequently characterized using a PCR-based method to identify the approximate location of the transposon insertion. Altogether, approximately 20 000 random mutants were screened and 51 different genes were identified as having potential involvement in the production of toxoflavin, lipase and/or extracellular polysaccharide. Especially, two regulatory genes, ntpR and tepR, encoding a LysR-type transcriptional regulator and a σ54-dependent response regulator, respectively, were discovered in this study as new negative regulatory factors for the production of toxoflavin, the major phytotoxin synthesized by B. glumae and involved in bacterial pathogenesis.

Diversities in virulence, antifungal activity, pigmentation and DNA fingerprint among strains of Burkholderia glumae.

Burkholderia glumae is the primary causal agent of bacterial panicle blight of rice. In this study, 11 naturally avirulent and nine virulent strains of B. glumae native to the southern United States were characterized in terms of virulence in rice and onion, toxofalvin production, antifungal activity, pigmentation and genomic structure. Virulence of B. glumae strains on rice panicles was highly correlated to virulence on onion bulb scales, suggesting that onion bulb can be a convenient alternative host system to efficiently determine the virulence of B. glumae strains. Production of toxoflavin, the phytotoxin that functions as a major virulence factor, was closely associated with the virulence phenotypes of B. glumae strains in rice. Some strains of B. glumae showed various levels of antifungal activity against Rhizoctonia solani, the causal agent of sheath blight, and pigmentation phenotypes on casamino acid-peptone-glucose (CPG) agar plates regardless of their virulence traits. Purple and yellow-green pigments were partially purified from a pigmenting strain of B. glumae, 411gr-6, and the purple pigment fraction showed a strong antifungal activity against Collectotrichum orbiculare. Genetic variations were detected among the B. glumae strains from DNA fingerprinting analyses by repetitive element sequence-based PCR (rep-PCR) for BOX-A1R-based repetitive extragenic palindromic (BOX) or enterobacterial repetitive intergenic consensus (ERIC) sequences of bacteria; and close genetic relatedness among virulent but pigment-deficient strains were revealed by clustering analyses of DNA fingerprints from BOX-and ERIC-PCR.

Dissection of quorum-sensing genes in Burkholderia glumae reveals non-canonical regulation and the new regulatory gene tofM for toxoflavin production.

Burkholderia glumae causes bacterial panicle blight of rice and produces major virulence factors, including toxoflavin, under the control of the quorum-sensing (QS) system mediated by the luxI homolog, tofI, and the luxR homolog, tofR. In this study, a series of markerless deletion mutants of B. glumae for tofI and tofR were generated using the suicide vector system, pKKSacB, for comprehensive characterization of the QS system of this pathogen. Consistent with the previous studies by other research groups, ΔtofI and ΔtofR strains of B. glumae did not produce toxoflavin in Luria-Bertani (LB) broth. However, these mutants produced high levels of toxoflavin when grown in a highly dense bacterial inoculum (∼ 10(11) CFU/ml) on solid media, including LB agar and King's B (KB) agar media. The ΔtofI/ΔtofR strain of B. glumae, LSUPB201, also produced toxoflavin on LB agar medium. These results indicate the presence of previously unknown regulatory pathways for the production of toxoflavin that are independent of tofI and/or tofR. Notably, the conserved open reading frame (locus tag: bglu_2g14480) located in the intergenic region between tofI and tofR was found to be essential for the production of toxoflavin by tofI and tofR mutants on solid media. This novel regulatory factor of B. glumae was named tofM after its homolog, rsaM, which was recently identified as a novel negative regulatory gene for the QS system of another rice pathogenic bacterium, Pseudomonas fuscovaginae. The ΔtofM strain of B. glumae, LSUPB286, produced a less amount of toxoflavin and showed attenuated virulence when compared with its wild type parental strain, 336gr-1, suggesting that tofM plays a positive role in toxoflavin production and virulence. In addition, the observed growth defect of the ΔtofI strain, LSUPB145, was restored by 1 µM N-octanoyl homoserine lactone (C8-HSL).