Phytophthora is a filamentous fungus-like microorganism, but belongs to the oomycetes, in the kingdom Stramenopila. Phytophthora species are notorious as plant destroyers, causing multibillion-dollar damage to agriculture and natural ecosystems worldwide annually. For a long time, genome editing has been unattainable in oomycetes, because of their extremely low rate of homologous recombination. The recent implementation of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system in the soybean pathogen Phytophthora sojae, an experimental model for oomycetes, has opened up a powerful new research capability for the oomycete community. Here, we describe a detailed protocol for CRISPR/Cas9-mediated genome editing in P. sojae, including single guide RNA (sgRNA) design and construction, efficient gene replacement, and mutant-screening strategies. This protocol should be generally applicable for most culturable oomycetes. We also describe an optimized transformation method that is useful for other Phytophthora spp. including P. capsici and P. parasitica. © 2017 by John Wiley & Sons, Inc.

The perception of Phytophthora sojae avirulence (Avr) gene products by corresponding soybean resistance (Rps) gene products causes effector triggered immunity. Past studies have shown that the Avr3a and Avr5 genes of P. sojae are genetically linked, and the Avr3a gene encoding a secreted RXLR effector protein was recently identified. We now provide evidence that Avr3a and Avr5 are allelic. Genetic mapping data from F2 progeny indicates that Avr3a and Avr5 co-segregate, and haplotype analysis of P. sojae strain collections reveal sequence and transcriptional polymorphisms that are consistent with a single genetic locus encoding Avr3a/5. Transformation of P. sojae and transient expression in soybean were performed to test how Avr3a/5 alleles interact with soybean Rps3a and Rps5. Over-expression of Avr3a/5 in a P. sojae strain that is normally virulent on Rps3a and Rps5 results in avirulence to Rps3a and Rps5; whereas silencing of Avr3a/5 causes gain of virulence in a P. sojae strain that is normally avirulent on Rps3a and Rps5 soybean lines. Transient expression and co-bombardment with a reporter gene confirms that Avr3a/5 triggers cell death in Rps5 soybean leaves in an appropriate allele-specific manner. Sequence analysis of the Avr3a/5 gene identifies crucial residues in the effector domain that distinguish recognition by Rps3a and Rps5.

Phytophthora sojae causes root and stem rot, one of the most devastating diseases of soybean worldwide. In Heilongjiang and Fujian provinces in China, serious cases of Phytophthora stem and root rot have occurred and caused heavy losses in the past several years. To determine the current population status of this pathogen, we investigated the pathogen's distribution, pathotypes, and metalaxyl sensitivity in both provinces. P. sojae was baited and isolated from 258 soil samples in both provinces using the soybean leaf bait method. The pathotypes of all isolates were characterized on 13 differential soybean cultivars using the hypocotyl slit inoculation method, and the sensitivity of all isolates to metalaxyl was tested in vitro. In all, 75 isolates were recovered from 75 fields in 33 counties; of these, 31 counties were in Heilongjiang Province and 2 counties were in Fujian Province. Thirty-five new pathotypes were identified compared with the previously defined races. Less than 5% of the isolates were virulent to cultivars with individual Rps genes 1a, 1c, or 1k. No metalaxyl-resistant isolates were found; the half maximal effective concentration values of all isolates ranged from 0.04 to 0.22 μg ml–1. These results suggest that effective management of the disease in both provinces can be accomplished through the use of resistant cultivars with Rps genes 1a, 1c, or 1k and the fungicide metalaxyl.

The effector gene Avr1b-1 of Phytophthora sojae determines the efficacy of the resistance gene Rps1b in soybean. The sequences of the Avr1b-1 locus in 34 Chinese isolates of P. sojae were obtained and analysed by polymerase chain reaction (PCR) and inverse PCR. Four different alleles and a complete deletion mutation of the Avr1b-1 gene were identified. Molecular analysis of the deletion breakpoints in the Avr1b-1 locus revealed that an 8-kb DNA sequence containing Avr1b-1 was deleted and a 12.7-kb DNA sequence was inserted at the same locus. A truncated transposase gene was found and five transposable elements were predicted in the inserted sequence, suggesting that the deletion of Avr1b-1 might be attributed to transposon movement. The transcription of Avr1b-1 was analysed in virulent isolates containing four alleles of Avr1b-1 by real-time reverse transcription-PCR. In all virulent isolates, only those isolates containing the second allele transcripted Avr1b-1.