Identification and pathogenicity analysis of Fusarium spp. on peach in China.

BACKGROUND: Vascular system is affected by diseases that can seriously damage plant health by inducing browning and death of branches. This study aimed to identify and analyze the pathogenicity of Fusarium spp. isolates obtained from diseased peach branches in several peach-producing areas of China. RESULTS: We obtained and confirmed nine Fusarium isolates based on morphological and molecular characteristics. Phylogenetic relationships using a combination of rDNA-internal transcribed spacer (ITS), elongation factor (EF)-1α, and mitochondrial small subunit (mtSSU) gene sequences were analyzed. GJH-Z1, GJH-6, and GJH-1 were identified as F. avenaceum; HYR-Z3, and ZLZT-6 as F. concentricum, HH-2020-G2, and HYTZ-4 as F. solani, GG-2020-1 as F. asiaticum, SYGZ-1 as F. equiseti. Through acupuncture comparison, the pathogenicity of F. equiseti (SYGZ-1) was highest amongst nine strains. Meanwhile, F. concentricum (HYR-Z3 and ZLZT-6), and F. solaini (HYTZ-4) had a higher level of pathogenicity as revealed by impregnation. CONCLUSIONS: Our study shed light on the findings that Fusarium spp. can inflict vascular bundle browning of peach plants. Our results will extend the understanding of pathogenic diseases in China's peach industry.

Wheat rhizosphere colonization by Bacillus amyloliquefaciens W10 and Pseudomonas protegens FD6 suppress soil and in planta abundance of the sharp eyespot pathogen Rhizoctonia cerealis.

AIMS: Develop quantitative assays (qPCR) to determine the wheat rhizosphere competence of inoculant strains Bacillus amyloliquefaciens W10 and Pseudomonas protegens FD6, and their suppressive efficacies against the sharp eyespot pathogen Rhizoctonia cerealis. METHODS AND RESULTS: Antimicrobial metabolites of strains W10 and FD6 decreased in vitro growth of R. cerealis. A qPCR assay for strain W10 was designed from a diagnostic AFLP fragment and the rhizosphere dynamics of both strains in wheat seedlings were compared by culture-dependent (CFU) and qPCR assays. The qPCR minimum detection limits for strains W10 and FD6 were log 3.04 and log 4.03 genome (cell) equivalents g-1 soil, respectively. Inoculant soil and rhizosphere abundance determined by CFU and qPCR were highly correlated (r > 0.91). In wheat bioassays, rhizosphere abundance of strain FD6 was up to 80-fold greater (P < 0.001) than strain W10 at 14 and 28 days postinoculation. Both inoculants reduced (P < 0.05) rhizosphere soil and root abundance of R. cerealis by up to 3-fold. CONCLUSIONS: Strain FD6 exhibited greater abundance in wheat roots and rhizosphere soil than strain W10 and both inoculants decreased the rhizosphere abundance of R. cerealis.

Molecular and Biological Characterization of Two New Species Causing Peach Shoot Blight in China.

Peach shoot blight (PSB), which kills shoots, newly sprouted leaf buds, and peach fruits, has gradually increased over the last 10 years and resulted in 30 to 50% of total production loss of the peach industry in China. Phomopsis amygdali has been identified as the common causal agent of this disease. In this study, two new species, Phomopsis liquidambaris (strain JW18-2) and Diaporthe eres (strain JH18-2), were also pathogens causing PSB, as determined through molecular phylogenetic analysis based on the sequences of the internal transcribed spacer (ITS) region, translation elongation factor 1-α (EF1-α) and beta-tubulin (TUB), and colony and conidial morphological characteristics. Biological phenotypic analysis showed that the colony growth rate of strain JW18-2 was faster than that of strains JH18-2 and ZN32 (one of the P. amygdali strains that we previously found and identified). All three strains produced α-conidia; however, JW18-2 could not produce ß-conidia on alfalfa decoction and Czapek media, and the ß-conidia produced by strain JH18-2 were shorter in length and thicker in width than those produced by strain ZN32. Pathogenicity tests showed that JW18-2 presented the strongest pathogenicity for peach fruits and twigs and was followed by strains JH18-2 and ZN32. The results shed light on the etiology of PSB and provide a warning that P. liquidambaris or D. eres might develop into dominant species after a few years while also potentially benefitting the development of effective disease control management strategies.

Rapid Detection of Peach Shoot Blight Caused by Phomopsis amygdali Utilizing a New Target Gene Identified from Genome Sequences Within Loop-Mediated Isothermal Amplification.

Peach shoot blight (PSB), caused by Phomopsis amygdali, is a serious threat to the healthy development of the peach industry and leads to 30 to 50% damage to peach production in southern China. In this study, loop-mediated isothermal amplification (LAMP) technology was used to detect the P. amygdali target of a gene of GME6801 that was unique in the whole genome of the pathogen compared with that of Diaporthe (Phomopsis) longicolla TWH P74, Fusarium graminearum PH-1, Colletotrichum gloeosporioides SMCG1 and Magnaporthe oryzae 70-15. Blast comparison of this gene sequence in NCBI database showed that no homologous sequences were found. Therefore, the gene sequence of GME6801 was used to design two pairs of LAMP primers and one pair of PCR primers. The results showed that both primer sets were specific to the 15 strains of P. amygdali, and the other 15 fungal strains presented negative reactions, similar to the control. In addition, 50 pg of genomic DNA of P. amygdali in a 25-µl reaction system could be detected by LAMP assay, which was 100 times more sensitive than PCR. Furthermore, the GME6801 LAMP assay was used to detect artificially inoculated twigs of the pathogen, disease twigs within significantly symptomatic PSB in the fields, and healthy twigs in the same orchard, with detection rates of 100, 75, and 20.8%, respectively. However, detection rates of conventional PCR were separately 100, 62.5, and 16.7%. The results indicated that GME6801-based LAMP could be used for P. amygdali detection as its specificity, sensitivity, and simplicity. This study provides a rapid experimental basis for the identification and prediction of P. amygdali that causes PSB and is beneficial for precise prevention and control of the disease.

Expression of the small cysteine-rich protein SCR96 from Phytophthora cactorum in mammalian cells: phytotoxicity and exploitation of its polyclonal antibody.

OBJECTIVE: We aimed to investigate the expression of a novel small cysteine-rich (SCR) effector protein SCR96 from the phytopathogenic oomycete Phytophthora cactorum in mammalian cells, its bioactivity and to exploit its polyclonal antibody. RESULTS: The gene encoding the SCR effector protein SCR96 was codon-optimized, custom-synthesized, cloned into pcDNA3.1(-) and overexpressed in human embryonic kidney (HEK) 293-6E cells. The recombinant protein SCR96 was prone to aggregation and purified with its monomer to homogeneity with a predicted molecular weight of 8.9 kDa. SCR96 exhibited strong phytotoxic activity on tomato seedlings at 24 h post treatment with 4.2 µg of the purified protein. An anti-SCR96 polyclonal antibody was prepared by immunization of New Zealand white rabbits. The good-titer antibody had a detection sensitivity at 6.25-ng level and could specifically detect the SCR96 protein expressed either in yeast, or in tomato leaves. CONCLUSIONS: Transient production of the SCR effector protein SCR96 in mammalian cells is reliable, providing sufficient recombinant protein that can be utilized for analysis of its phytotoxic activity and preparation of its polyclonal antibody.

Induced resistance in nectarine fruit by Bacillus licheniformis W10 for the control of brown rot caused by Monilinia fructicola.

Brown rot caused by Monilinia fructicola has led to considerable preharvest and postharvest losses in all major nectarine fruit-growing areas. In our previous study, we successfully identified a biocontrol strain of bacteria, Bacillus licheniformis W10, that can be used to control brown rot. However, the possible mechanism of the control of brown rot by B. licheniformis W10 is still unclear. Therefore, the objectives of this study were to determine whether B. licheniformis W10 induces resistance by activating defense-related enzymes including antioxidant enzymes in nectarine. Treatment of nectarine fruit with B. licheniformis W10 reduced both M. fructicola-induced oxidative damage and reactive oxygen species (ROS) production. Furthermore, application of B. licheniformis to nectarine fruit resulted in a significant increase in the activity of antioxidant and defense-related enzymes and increase in the expression of the corresponding genes. Overall, our results verified the proposed mechanism of B. licheniformis W10 in controlling M. fructicola via regulation of ROS levels and activation of antioxidant and defense-related enzymes.

Coiled-Coil N21 of Hpa1 in Xanthomonas oryzae pv. oryzae Promotes Plant Growth, Disease Resistance and Drought Tolerance in Non-Hosts via Eliciting HR and Regulation of Multiple Defense Response Genes.

Acting as a typical harpin protein, Hpa1 of Xanthomonas oryzae pv. oryzae is one of the pathogenic factors in hosts and can elicit hypersensitive responses (HR) in non-hosts. To further explain the underlying mechanisms of its induced resistance, we studied the function of the most stable and shortest three heptads in the N-terminal coiled-coil domain of Hpa1, named N21Hpa1. Proteins isolated from N21-transgenic tobacco elicited HR in Xanthi tobacco, which was consistent with the results using N21 and full-length Hpa1 proteins expressed in Escherichia coli. N21-expressing tobacco plants showed enhanced resistance to tobacco mosaic virus (TMV) and Pectobacterium carotovora subsp. carotovora (Pcc). Spraying of a synthesized N21 peptide solution delayed the disease symptoms caused by Botrytis cinerea and Monilinia fructicola and promoted the growth and drought tolerance of plants. Further analysis indicated that N21 upregulated the expression of multiple plant defense-related genes, such as genes mediated by salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signaling, and genes related to reactive oxygen species (ROS) biosynthesis. Further, the bioavailability of N21 peptide was better than that of full-length Hpa1Xoo. Our studies support the broad application prospects of N21 peptide as a promising succedaneum to biopesticide Messenger or Illite or other biological pharmaceutical products, and provide a basis for further development of biopesticides using proteins with similar structures.

Pyridoxine biosynthesis protein MoPdx1 affects the development and pathogenicity of Magnaporthe oryzae.

B vitamins are essential micro-organic compounds for the development of humans and animals. Vitamin B6 comprises a group of components including pyridoxine, pyridoxal, and pyridoxamine. In addition, vitamin B6 acts as the coenzymes in amino acid biosynthesis, decarboxylation, racemic reactions, and other biological processes. In this study, we found that the expressions of a gene encoding pyridoxine biosynthesis protein (PDX1) were significantly upregulated in the early infectious stages in M. oryzae. Furthermore, deletion of MoPDX1 slowed vegetative growth on different media, especially on MM media, and the growth defect was rescued when MoPdx1-protein was expressed in mutants strains and when commercial VB6 (pyridoxine) was added exogenously. However, VB6 content in different strains cultured in CM media has no significant difference, suggested that MoPdx1 was involved in de novo VB6 biosynthesis not in uptake process, and VB6 regulates the vegetative growth of M. oryzae. The ΔMopdx1 mutants presented abnormal appressorium turgor, slowed invasive growth and reduced virulence on rice seedlings and sheath cells. MoPdx1 was located in the cytoplasm and present in spore and germ tubes at 14 hours post inoculation (hpi) and then transferred into the appressorium at 24 hpi. Addition of VB6 in the conidial suspentions could rescue the defects of appressorium turgor pressure at 14 hpi or 24 hpi, invasive growth and pathogenicity of the MoPDX1 deletion mutants. Indicated that MoPdx1 affected the appressorium turgor pressure, invasive growth and virulence mainly depended on de novo VB6, and VB6 was biosynthesized in conidia, then transported into the appressorium, which play important roles in substances transportation from conidia to appressorium thus to regulate the appressorium turgor pressure. However, deletion of MoPDX1 did not affect the ability that scavenge ROS produced by rice cells, and the mutant strains were unable to activate host defense responses. In addition, co-immunoprecipitation (Co-IP) assays investigating potential MoPdx1-interacting proteins suggested that MoPdx1 might take part in multiple pathways, especially in the ribosome and in biosynthesis of some substances. These results indicate that vitamins are involved in the development and pathogenicity of M. oryzae.

Integrative transcriptomic and proteomic analyses reveal a positive role of BES1 in salt tolerance in Arabidopsis.

Introduction: Salt stress is a major environmental factor limiting plant growth and development. Previous studies have indicated that the steroidal hormones-brassinosteroids (BRs) are important regulators of plant responses to salt stress. However, the underlying molecular mechanisms have not been fully understood. Methods: (1) Phenotypic analysis of bes1-D, BES1-RNAi and their wild-type (Col-0) under salt treatments with different concentrations of NaCl. (2) Transcriptomic and proteomic profiling of BES1-regulated genes and proteins under salt treatment; (3) qRT-PCR validation of selected BES1-regulated genes under salt stress; (4) Transient transcriptional assay of BES1 regulation on its putative target genes in Arabidopsis protoplasts; (5) Electrophoresis Mobility Shift Assay (EMSA) of BES1 binding with its potential target genes. Results and Discussion: Phenotypic analysis indicated that bes1-D, a gain-of-function mutant of the BR-regulated transcription factor BES1 in Arabidopsis showed better salt tolerance than the wild-type plant, while a BES1 RNA interference (BES1-RNAi) line was more sensitive to salt stress. Global gene expression profiling and time series clustering analyses identified a total of 1,170 genes whose expression was boosted in bes1-D under salt stress. Further GO enrichment and gene functional network analyses identified several key modules that are regulated by BES1 and most sensitive to salt stress perturbations, including stress response, response to ABA and ROS, flavonoid biosynthesis and transmembrane transport. A comparative proteomic analysis performed under the same stress conditions supported the results from the transcriptome analysis. In addition, transient gene transcription assays in Arabidopsis protoplasts and in vitro DNA binding assays verified that BES1 regulates the expression of some ion transporter genes directly and indirectly. Taken together, our results support a positive role of BES1 in plant salt tolerance.

Broad-spectrum resistance mechanism of serine protease Sp1 in Bacillus licheniformis W10 via dual comparative transcriptome analysis.

Antagonistic microorganisms are considered to be the most promising biological controls for plant disease. However, they are still not as popular as chemical pesticides due to complex environmental factors in the field. It is urgent to exploit their potential genetic characteristics and excellent properties to develop biopesticides with antimicrobial substances as the main components. Here, the serine protease Sp1 isolated from the Bacillus licheniformis W10 strain was confirmed to have a broad antifungal and antibacterial spectrum. Sp1 treatment significantly inhibited fungal vegetative growth and damaged the structure of hyphae, in accordance with that caused by W10 strain. Furthermore, Sp1 could activate the systemic resistance of peach twigs, fruits and tobacco. Dual comparative transcriptome analysis uncovered how Sp1 resisted the plant pathogenic fungus Phomopsis amygdali and the potential molecular resistance mechanisms of tobacco. In PSp1 vs. P. amygdali, RNA-seq identified 150 differentially expressed genes (DEGs) that were upregulated and 209 DEGs that were downregulated. Further analysis found that Sp1 might act on the energy supply and cell wall structure to inhibit the development of P. amygdali. In TSp1 vs. Xanthi tobacco, RNA-seq identified that 5937 DEGs were upregulated and 2929 DEGs were downregulated. DEGs were enriched in the metabolic biosynthesis pathways of secondary metabolites, plant hormone signal transduction, plant-pathogen interactions, and MAPK signaling pathway-plant and further found that the genes of salicylic acid (SA) and jasmonic acid (JA) signaling pathways were highly expressed and the contents of SA and JA increased significantly, suggesting that systemic resistance induced by Sp1 shares features of SAR and ISR. In addition, Sp1 might induce the plant defense responses of tobacco. This study provides insights into the broad-spectrum resistance molecular mechanism of Sp1, which could be used as a potential biocontrol product.

Two coiled-coil regions of Xanthomonas oryzae pv. oryzae harpin differ in oligomerization and hypersensitive response induction.

Hpa1(Xoo) (harpin) is a type III secreted protein of the rice blight bacterial pathogen Xanthomonas oryzae pv. oryzae that elicits a hypersensitive response (HR) in nonhost tobacco. Hpa1(Xoo) is predicted to contain two potential coiled-coil (CC) regions, one at the N-terminus with a high probability of formation, and one at the C-terminus with a lower probability of formation. We constructed several CC-equivalent peptides by a chemosynthetic method, and investigated the structure-function of the predicted Hpa1(Xoo) CC regions, using biophysical and biochemical approaches. Both peptides elicited an HR in tobacco. Mutant versions of the N- and C-terminal peptides that were predicted to disrupt or favor CC formation were generated. The resulting altered HR activity and oligomerization indicated that the N-terminal CC region is essential for eliciting HR, but the C-terminus is not. The results also indicate that a 14-residue fragment (LDQLLCQLISALLQ) within the N-terminal CC region is a minimal and independent functional element for HR-induction in tobacco leaves. We propose that HR-induction requires a specific oligomerization of the CC regions of Hpa1(Xoo).

Glutathione contributes to resistance responses to TMV through a differential modulation of salicylic acid and reactive oxygen species.

Systemic acquired resistance (SAR) is induced by pathogens and confers protection against a broad range of pathogens. Several SAR signals have been characterized, but the nature of the other unknown signalling by small metabolites in SAR remains unclear. Glutathione (GSH) has long been implicated in the defence reaction against biotic stress. However, the mechanism that GSH increases plant tolerance against virus infection is not entirely known. Here, a combination of a chemical, virus-induced gene-silencing-based genetics approach, and transgenic technology was undertaken to investigate the role of GSH in plant viral resistance in Nicotiana benthamiana. Tobacco mosaic virus (TMV) infection results in increasing the expression of GSH biosynthesis genes NbECS and NbGS, and GSH content. Silencing of NbECS or NbGS accelerated oxidative damage, increased accumulation of reactive oxygen species (ROS), compromised plant resistance to TMV, and suppressed the salicylic acid (SA)-mediated signalling pathway. Application of GSH or l-2-oxothiazolidine-4-carboxylic acid (a GSH activator) alleviated oxidative damage, decreased accumulation of ROS, elevated plant local and systemic resistance, enhanced the SA-mediated signalling pathway, and increased the expression of ROS scavenging-related genes. However, treatment with buthionine sulfoximine (a GSH inhibitor) accelerated oxidative damage, elevated ROS accumulation, compromised plant systemic resistance, suppressed the SA-mediated signalling pathway, and reduced the expression of ROS-regulating genes. Overexpression of NbECS reduced oxidative damage, decreased accumulation of ROS, increased resistance to TMV, activated the SA-mediated signalling pathway, and increased the expression of the ROS scavenging-related genes. We present molecular evidence suggesting GSH is essential for both local and systemic resistance of N. benthamiana to TMV through a differential modulation of SA and ROS.

Identification and characterization of a serine protease from Bacillus licheniformis W10: A potential antifungal agent.

Bacillus licheniformis W10 is a strain of biocontrol bacteria that was obtained from plant rhizosphere screening. In this study, we purified, identified, and carried out bioinformatics analysis of the W10 antifungal protein from Bacillus licheniformis. Mass spectrometry analysis was carried out by passing the antifungal protein through a high-resolution time-of-flight mass spectrometer. Mascot searches of the tandem mass spectrometry data identified this antifungal protein as a serine protease, and the 1347 bp gene encoding this protein was cloned. Bioinformatics analysis of this protein indicated that it contains 448 amino acid residues, has a molecular weight of 48,794.16 Da and an isoelectric point of 6.04, and is a hydrophilic protein. In the secondary and tertiary structure of this protein, the proportion of α-helices and ß-folds is similar, and the protein possesses a Peptidase_S8 conserved domain. Using BApNA as a substrate, it was found that the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) can inhibit the W10 antifungal protein. PMSF concurrently reduced the inhibitory effects of the antifungal protein on Botrytis cinerea, showing that the W10 antifungal protein possesses serine protease activity. The W10 antifungal protein has good thermal stability. The study implies potential of this enzyme for biocontrol of fungal plant pathogens.

Alpha-momorcharin enhances Nicotiana benthamiana resistance to tobacco mosaic virus infection through modulation of reactive oxygen species.

Alpha-momorcharin (α-MMC), a member of the plant ribosomal inactivating proteins (RIPs) family, has been proven to exhibit important biological properties in animals, including antiviral, antimicrobial, and antitumour activities. However, the mechanism by which α-MMC increases plant resistance to viral infections remains unclear. To study the effect of α-MMC on plant viral defence and how α-MMC increases plant resistance to viruses, recombinant DNA and transgenic technologies were employed to investigate the role of α-MMC in Nicotiana benthamiana resistance to tobacco mosaic virus (TMV) infection. Treatment with α-MMC produced through DNA recombinant technology or overexpression of α-MMC mediated by transgenic technology alleviated TMV-induced oxidative damage and reduced the accumulation of reactive oxygen species (ROS) during TMV-green fluorescent protein infection of N. benthamiana. There was a significant decrease in TMV replication in the upper leaves following local α-MMC treatment and in α-MMC-overexpressing plants relative to control plants. These results suggest that application or overexpression of α-MMC in N. benthamiana increases resistance to TMV infection. Finally, our results showed that overexpression of α-MMC up-regulated the expression of ROS scavenging-related genes. α-MMC confers resistance to TMV infection by means of modulating ROS homeostasis through controlling the expression of antioxidant enzyme-encoding genes. Overall, our study revealed a new crosstalk mechanism between α-MMC and ROS during resistance to viral infection and provides a framework to understand the molecular mechanisms of α-MMC in plant defence against viral pathogens.

Simultaneous silencing of two target genes using virus-induced gene silencing technology in Nicotiana benthamiana.

Virus-induced gene silencing (VIGS) is an effective strategy for rapid gene function analysis. It is well established that the NAC transcription factor and salicylic acid (SA) signal pathway play essential roles in response to biotic stresses. However, simultaneous silencing of two target genes using VIGS in plants has been rarely reported. Therefore, in this report, we performed VIGS to silence simultaneously the SA-binding protein 2 (NbSABP2) and NbNAC1 in Nicotiana benthamiana to investigate the gene silencing efficiency of simultaneous silencing of two genes. We first cloned the full-length NbNAC1 gene, and the characterization of NbNAC1 was also analysed. Overlap extension polymerase chain reaction (PCR) analysis showed that the combination of NbSABP2 and NbNAC1 was successfully amplified. Bacteria liquid PCR confirmed that the combination of NbSABP2 and NbNAC1 was successfully inserted into the tobacco rattle virus vector. The results showed that the leaves from the NbSABP2 and NbNAC1 gene-silenced plants collapsed slightly, with browning at the base of petiole or veina. Quantitative real-time PCR results showed that the expression of NbSABP2 and NbNAC1 were significantly reduced in 12 days post silenced plants after tobacco rattle virus infiltration compared with the control plants. Overall, our results suggest that VIGS can be used to silence simultaneously two target genes.

Mutations in the N-terminal coding region of the harpin protein Hpa1 from Xanthomonas oryzae cause loss of hypersensitive reaction induction in tobacco.

Harpins encoded by many gram-negative phytopathogenic bacterial hrp genes induce hypersensitive response (HR) and associated defense responses on nonhost plants. Hpa1(Xoo) and Hpa1(Xoc), two harpin proteins from Xanthomonas oryzae pathovars, induce HR when infiltrated into tobacco leaves. N- and C-terminal mutations of Hpa1(Xoo) and Hpa1(Xoc), respectively, were tested for their ability to elicit HR on tobacco. Deletion of codons for 12 highly hydrophilic amino acids (H(2)N-QGISEKQLDQLL-COOH) that partially overlap the N-terminal alpha-helical regions of respective proteins was found to be critical for the elicitation of HR in tobacco. Furthermore, two single missense mutants Hpa1(Xoo) (L51P) and Hpa1(Xoc) (L53P) that are predicted to destroy the coiled-coil integrity and inhibit the dimer formation eliminated HR elicitation activity in tobacco. However, both wild-type proteins and derivative mutants retained the ability to induce systemic acquired resistance in tobacco against tobacco mosaic virus. Accumulations of npr1 (nonexpressor of pathogenesis-related protein 1), hsr515 (hypersensitivity-related protein 515), and pr2 (pathogenesis-related protein 2) transcripts were found in tobacco plants infiltrated with wild-type or mutated proteins.

The Plant Ribosome-Inactivating Proteins Play Important Roles in Defense against Pathogens and Insect Pest Attacks.

Ribosome-inactivating proteins (RIPs) are toxic N-glycosidases that depurinate eukaryotic and prokaryotic rRNAs, thereby arresting protein synthesis during translation. RIPs are widely found in various plant species and within different tissues. It is demonstrated in vitro and in transgenic plants that RIPs have been connected to defense by antifungal, antibacterial, antiviral, and insecticidal activities. However, the mechanism of these effects is still not completely clear. There are a number of reviews of RIPs. However, there are no reviews on the biological functions of RIPs in defense against pathogens and insect pests. Therefore, in this report, we focused on the effect of RIPs from plants in defense against pathogens and insect pest attacks. First, we summarize the three different types of RIPs based on their physical properties. RIPs are generally distributed in plants. Then, we discuss the distribution of RIPs that are found in various plant species and in fungi, bacteria, algae, and animals. Various RIPs have shown unique bioactive properties including antibacterial, antifungal, antiviral, and insecticidal activity. Finally, we divided the discussion into the biological roles of RIPs in defense against bacteria, fungi, viruses, and insects. This review is focused on the role of plant RIPs in defense against bacteria, fungi, viruses, and insect attacks. The role of plant RIPs in defense against pathogens and insects is being comprehended currently. Future study utilizing transgenic technology approaches to study the mechanisms of RIPs will undoubtedly generate a better comprehending of the role of plant RIPs in defense against pathogens and insects. Discovering additional crosstalk mechanisms between RIPs and phytohormones or reactive oxygen species (ROS) against pathogen and insect infections will be a significant subject in the field of biotic stress study. These studies are helpful in revealing significance of genetic control that can be beneficial to engineer crops tolerance to biotic stress.