Novel Factors from Phytophthora palmivora Promote Its Infection and a Potential Elicitor from Acanthophora spicifera Enhances Hevea brasiliensis Resistance

Abstract

Phytophthora palmivora is a destructive oomycete plant pathogen with a wide host range. So far, little is known about the factors governing its infection structure development and pathogenicity. From the culture filtrate of P. palmivora, I identified a secreted glycoprotein of 15 kDa, designated as Ppal15kDa, using liquid chromatography tandem mass spectrometry (LC-MS/MS). Two variants, Ppal15kDaA and Ppal15kDaB were amplified from a P. palmivora papaya isolate. Transient expression of both variants in Nicotiana benthamiana by agroinfiltration enhanced P. palmivora infection. Six Ppal15kDa mutants with diverse mutations were generated via CRISPR/Cas9-mediated gene editing. All mutants were compromised in infectivity on N. benthamiana and papaya. Two of the mutants which all Ppal15kDa copies were mutated lost almost complete pathogenicity. The pathogenicity of the other four containing at least one wild-type copy of Ppal15kDa was compromised at varying levels. The mutants were also affected in development as they produced smaller sporangia, shorter germ tubes, and less appressoria. Interestingly, the affected levels in development corresponded to the levels of reduction in pathogenicity, suggesting that Ppal15kDa plays an important role in normal development of P. palmivora infection structures, which contributes to successful infection. Consistent with its role in infection structure development and pathogenicity, Ppal15kDa was found to be highly induced during appressorium formation. In addition, Ppal15kDa homologs are broadly present in Phytophthora spp, but none were characterized. Altogether, this study identified a novel component involved in development and pathogenicity of P. palmivora and possibly many other Phytophthora spp. Elicitors play an important role in plant and pathogen interactions. The discovery of new elicitors and their effects on plant defense responses is significant and challenging. In this study, I investigated novel elicitors from P. palmivora and their effects on plant defenses. A crude elicitor isolated by ethanol precipitation from culture filtrates of P. palmivora induced cell death in tobacco leaves. When tobacco leaves were infiltrated with this cell death-inducing elicitor, the accumulations of H2O2, salicylic acid (SA), scopoletin (Scp) and abscisic acid (ABA) were detected. Accumulations of SA, Scp and ABA were also induced in rubber tree leaves. P. palmivora infection significantly increased in rubber tree leaves pretreated with the elicitor and co-treated with the elicitor and zoospores of P. palmivora. This elicitor can be described as compound elicitor because Fourier-transform infrared (FTIR) spectroscopy revealed that it consisted of both polysaccharides and proteins. I also found that the cell death effect caused by this compound elicitor was completely neutralized by Proteinase K. The compound elicitor was composed of four fractions which were beta-glucan (F1), high-molecular-weight glycoprotein (F2), broad- molecular-weight glycoprotein (F3) and 42 kDa protein (F4). Interestingly, the broad- molecular-weight glycoprotein (F3) caused the highest level of cell death in tobacco leaves, while the beta-glucan (F1) had no effect. The high-molecular-weight glycoprotein (F2), broad-molecular-weight glycoprotein (F3) and 42 kDa protein (F4) fractions not only caused cell death in tobacco leaves but also induced high levels of SA accumulation. Furthermore, these three fractions clearly promoted P. palmivora infection of rubber tree leaves. P. palmivora is a hemibiotrophic pathogen. It can survive in dead tissue. This study revealed that P. palmivora could use cell death- inducing elicitors to promote its infection. Elicitors from seaweeds are considered as alternative stimulants of plant defenses against pathogenic infection. Finding new sources of elicitors and exploring their effects on plant defenses is a significant undertaking. In this study, I extracted crude polysaccharide (CPS) from Acanthophora spicifera (a red alga) and tested the effects of the compound on rubber tree (Hevea brasiliensis) defense responses. Accumulations of SA and Sep were measured by HPLC. The expressions of SA- and Jasmonic acid (JA)-responsive genes were analyzed by semi-qRT-PCR. Strong anion exchange chromatography and FTIR spectroscopy were used for purification and functional characterization of CPS, respectively. The extracted CPS enhanced rubber tree defenses against P. palmivora infection. It induced SA and Scp accumulations and SA-responsive gene expression, but suppressed JA-responsive gene expression. I successfully separated the non-sulphated polysaccharide (F1) from the sulphated polysaccharides (SPS). Both peaks of SPS (F2 and F3) were identified as lambda (2)-carrageenan. The F3 fraction showed greater elicitor activity on tobacco leaves. It induced SA and Sep accumulations and peroxidase activity but suppressed catalase activity. Furthermore, the purified 2-carrageenan did not cause cell death in tobacco or rubber tree leaves. Therefore, the elicitor from A. spicifera could be an alternative plant stimulant.