The Mevalonate Pathway Is Important for Growth, Spore Production, and the Virulence of Phytophthora sojae

The mevalonate (MVA) pathway in eukaryotic organisms produces isoprenoids, sterols, ubiquinone, and dolichols. These molecules are vital for diverse cellular functions, ranging from signaling to membrane integrity, and from post-translational modification to energy homeostasis. However, information on the MVA pathway in Phytophthora species is limited. In this study, we identified the MVA pathway genes and reconstructed the complete pathway in Phytophthora sojae in silico. We characterized the function of the MVA pathway of P. sojae by treatment with enzyme inhibitor lovastatin, deletion of the geranylgeranyl diphosphate synthase gene (PsBTS1), and transcriptome profiling analysis. The MVA pathway is ubiquitously conserved in Phytophthora species. Under lovastatin treatment, mycelial growth, spore production, and virulence of P. sojae were inhibited but the zoospore encystment rate increased. Heterozygous mutants of PsBTS1 showed slow growth, abnormal colony characteristics, and mycelial morphology. Mutants showed decreased numbers of sporangia and oospores as well as reduced virulence. RNA sequencing analysis identified the essential genes in sporangia formation were influenced by the enzyme inhibitor lovastatin. Our findings elucidate the role of the MVA pathway in P. sojae and provide new insights into the molecular mechanisms underlying the development, reproduction, and virulence of P. sojae and possibly other oomycetes. Our results also provide potential chemical targets for management of plant Phytophthora diseases.

A novel Phytophthora sojae effector PsFYVE1 modulates transcription and alternative splicing of immunity related genes by targeting host RZ-1A protein

Oomycete pathogens secrete many effectors to manipulate plant immunity and promote infection. However, relatively few effector types have been well characterized. In this study, members of a FYVE domain-containing protein family that is highly expanded in oomycetes were systematically identified, and one secreted protein, PsFYVE1, was selected for further study. PsFYVE1 enhanced Phytophthora infection in Nicotiana benthamiana and was necessary for P. sojae virulence. The FYVE domain of PsFYVE1 had PI3P-binding activity that depended on four conservative amino acid residues. Furthermore, PsFYVE1 targeted RNA-binding proteins RZ-1A/1B/1C in N. benthamiana and soybean, and silencing of NbRZ-1A/1B/1C genes attenuates plant immunity. NbRZ-1A was associated with spliceosome that included three important components, NbGRP7, NbGRP8, and NbU1-70K. Notably, PsFYVE1 could disrupt NbRZ-1A–NbGRP7 interaction. RNA-seq and subsequent experimental analysis demonstrated that PsFYVE1 and NbRZ-1A not only co-regulated transcription of NbHCT, NbEIN2, and NbSUS4 genes but also modulated pre-mRNA alternative splicing (AS) of the NbNSL1 gene, which participated in plant immunity. Collectively, these findings indicate that the FYVE domain-containing protein family includes potential new effector types and also highlight that plant pathogen effectors can regulate plant immunity related genes at both transcription and AS levels to promote disease.Author summaryMany plant pathogenic oomycetes secrete effector proteins into plants to facilitate infection. Discovering potential repertoire of novel effectors and corresponding molecular mechanisms are major themes in the study of oomycete–plant interactions. Here, we characterized a FYVE domain-containing protein (PsFYVE1) in P. sojae. PsFYVE1 carries a functional secretory signal peptide and is a virulence-essential effector for P. sojae infection. We demonstrated that PsFYVE1 interacted with a class of plant RNA-binding proteins, including soybean GmRZ-1A/1B/1C and N. benthamiana NbRZ-1A/1B/1C. Silencing of NbRZ-1A/1B/1C proteins increased Phytophthora infection and suppressed plant defense. Furthermore, NbRZ-1A interacted with the spliceosome components, and PsFYVE1 disrupted association between NbRZ-1A and spliceosome component NbGRP7. We examined the global transcription and alternative splicing (AS) changes regulated by PsFYVE1 and NbRZ-1A, which indicated that PsFYVE1 and NbRZ-1A co-regulated transcription and pre-mRNA AS of immunity-related genes. Thus, this study identifies a novel virulence-related effector from P. sojae and a class of positive regulators of plant immunity, and reveals a detailed mechanism of effector-medicated transcription and AS regulation during pathogen–plant interactions.

An atypical Phytophthora sojae RxLR effector manipulates host vesicle trafficking to promote infection

In plants, the apoplast is a critical battlefield for plant-microbe interactions. Plants secrete defense-related proteins into the apoplast to ward off the invasion of pathogens. How microbial pathogens overcome plant apoplastic immunity remains largely unknown. In this study, we reported that an atypical RxLR effector PsAvh181 secreted by Phytophthora sojae, inhibits the secretion of plant defense-related apoplastic proteins. PsAvh181 localizes to plant plasma membrane and essential for P. sojae infection. By co-immunoprecipitation assay followed by liquid chromatography-tandem mass spectrometry analyses, we identified the soybean GmSNAP-1 as a candidate host target of PsAvh181. GmSNAP-1 encodes a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein, which associates with GmNSF of the SNARE complex functioning in vesicle trafficking. PsAvh181 binds to GmSNAP-1 in vivo and in vitro. PsAvh181 interferes with the interaction between GmSNAP-1 and GmNSF, and blocks the secretion of apoplastic defense-related proteins, such as pathogenesis-related protein PR-1 and apoplastic proteases. Taken together, these data show that an atypical P. sojae RxLR effector suppresses host apoplastic immunity by manipulating the host SNARE complex to interfere with host vesicle trafficking pathway.

Mutations in the Promoter and Coding Regions of Avr3a Cause Gain of Virulence of Phytophthora sojae to Rps3a in Soybean

Phytophthora sojae threatens soybean production worldwide, and the cultivation of soybean cultivars carrying Rps genes is the most effective way to control this pathogen. However, DNA mutations in the Avr genes of P. sojae can escape recognization of the corresponding Rps genes, leading to the loss of soybean resistance. In this study, we investigated sequence polymorphism and transcript level of the Avr3a gene in Chinese isolates of P. sojae. Twenty-four mutations resulting in five unique Avr3a alleles were discovered in the Avr3a coding region from 32 P. sojae isolates. The Avr3a transcripts were detectable in the isolates containing Avr3a(I), Avr3a(II), Avr3a(III), and Avr3a(IV) but not in the isolates containing Avr3a(V). Promoter and 5'-UTR sequence analysis revealed eight unique mutations in the promoter region of Avr3a(V), suggesting that the mutations could result in the loss of Avr3a(V) transcription. Virulence tests indicated the isolates containing Avr3a(II) and Avr3a(IV) were virulent, suggesting that the mutations in the coding regions of Avr3a(II) and Avr3a(IV) caused the gain of virulence to Rps3a. Based on DNA mutations of Avr3a in virulent alleles, two SNP markers and one PCR-based marker were developed successfully for detecting the virulence of P. sojae isolates to Rps3a. These findings provide new insights into escape mechanisms of Avr3a and effective support for accurate pathotype identification of P. sojae using molecular methods.

Paenibacillus sp. strain UY79, isolated from a root nodule of Arachis villosa , displays a broad spectrum of antifungal activity

A nodule-inhabiting Paenibacillus sp. strain (UY79) isolated from wild peanut ( Arachis villosa ) was screened for its antagonistic activity against diverse fungi and oomycetes ( Botrytis cinerea , Fusarium verticillioides , Fusarium oxysporum , Fusarium graminearum , Fusarium semitectum , Macrophomina phaseolina , Phomopsis longicolla , Pythium ultimum, Phytophthora sojae, Rhizoctonia solani , Sclerotium rolfsii and Trichoderma atroviride ). Results obtained show that Paenibacillus sp. UY79 was able to antagonize these fungi/oomycetes and that agar-diffusible compounds and volatile compounds (different from HCN), participate in the antagonism exerted. Acetoin, 2,3-butanediol and 2-methyl-1-butanol were identified among the volatile compounds produced by UY79 strain with possible antagonistic activity against fungi/oomycetes. Paenibacillus sp. strain UY79 did not affect symbiotic association or growth promotion of alfalfa plants when co-inoculated with rhizobia. By whole genome sequence analysis, we determined that strain UY79 is a new species of Paenibacillus within the Paenibacillus polymyxa complex. Diverse genes putatively involved in biocontrol activity were identified in the UY79 genome. Furthermore, according to genome mining and antibiosis assays, strain UY79 would have the capability to modulate the growth of bacteria commonly found in soil/plant communities. IMPORTANCE Phytopathogenic fungi and oomycetes are responsible for causing devastating losses in agricultural crops. Therefore, there is an enormous interest in the development of effective and complementary strategies that allow the control of the phytopathogens, reducing the input of agrochemicals in croplands. Discovery of new strains with expanded antifungal activities and with a broad spectrum of action is challenging and of great future impact. Diverse strains belonging to the P. polymyxa complex have been reported to be effective biocontrol agents. Results presented here show that the novel discovered strain of Paenibacillus sp. presents diverse traits involved in antagonistic activity against a broad spectrum of pathogens and would be a potential and valuable strain to be further assessed for the development of biofungicides.

A giant NLR gene confers broad-spectrum resistance to Phytophthora sojae in soybean

Phytophthora root and stem rot caused by P. sojae is a destructive soybean soil-borne disease found worldwide. Discovery of genes conferring broad-spectrum resistance to the pathogen is a need to prevent the outbreak of the disease. Here, we show that soybean Rps11 is a 27.7-kb nucleotide-binding site-leucine-rich repeat (NBS-LRR or NLR) gene conferring broad-spectrum resistance to the pathogen. Rps11 is located in a genomic region harboring a cluster of large NLR genes of a single origin in soybean, and is derived from rounds of unequal recombination. Such events result in promoter fusion and LRR expansion that may contribute to the broad resistance spectrum. The NLR gene cluster exhibits drastic structural diversification among phylogenetically representative varieties, including gene copy number variation ranging from five to 23 copies, and absence of allelic copies of Rps11 in any of the non-Rps11-donor varieties examined, exemplifying innovative evolution of NLR genes and NLR gene clusters.