Rice Lesion Mimic Mutant (LMM) 6321-2 Exhibits Resistance Against Rice Blast ( Magnaporthe oryzae ) via Increased
Hypersensitive Response
Joseph Martin Q. Paet1*, Bo Zhou2, Mary Jeanie Telebanco-Yanoria3 and Rina B. Opulencia4
1 Bicol University Research and Development Management Division, Legazpi City, Philippines
2 Food and Agriculture Organization of the United Nations
3 International Rice and Research Institute, Laguna, Philippines
4 University of the Philippines Los Baños, Laguna, Philippines Corresponding author: [email protected]
Abstract
Hypersensitive response (HR) is a key feature of plant immunity that sacrifices through programmed cell death (PCD) the already infected cells and thereby restricts the growth and further invasion of pathogens. Rice (Oryza sativa) lesion mimic mutants (LMM) manifest disease phenotype in the absence of pathogen and are monocots’ model organisms in unraveling the molecular mechanism of HR-induced PCD. The LMM 6321-2 used in this study was from an ethyl methanesulfonate-generated mutant accession obtained from IRRI, Philippines and was phenotypically and genetically characterized. It was an initiator type of LMM with significantly reduced agronomic yield potential. However, LMM 6321-2 exhibited increased or partial resistance against virulent rice blast fungus, Magnaporthe oryzae (PO6-6 and CA89 isolates) and induced HR-mediated PCD after avirulent isolate (V86010, CA41 and M101- 1-2-9-1) inoculation using rice blast nursery spray and punch inoculation assays. Segregation analysis using the F2 population showed LMM 6321-2 to be a single recessive mutation. Expression profiling of PCD-associated genes showed significant changes in its expression profile both in non-inoculated set- up and M. oryzae post-inoculated experiments where genes of NADPH oxidases, reactive oxygen species (ROS) scavengers, LMM-, phytohormone-, senescence- and pathogenesis-related genes were found to be significantly expressed with a different timing of expression than its IR64 parent. In brief, the mutant showed delayed expression of PCD-related genes peaking 48 hours post-inoculation (hpi) compared to IR64, which heightened 24 hpi. Taken together, it was hypothesized that LMM 6321-2 have a ROS- mediated PCD where the mutation plays a key role in the lesion mimicking phenotype.
Keywords: gene expression, programmed cell death, resistance, ROS Introduction
Programmed cell death (PCD) is a complex and highly coordinated biological process of cellular suicide essential to the growth and development of higher forms of organisms. While it was well documented in animals generally as apoptosis, knowledge of PCD in plants remains a tera incognita, mainly because there are no clear homologous key regulators between the two kingdoms (Daneva, Gao, Durme, and Nowack, 2016; Taylor, Lam, and Lam, 2008). Furthermore, a relatively small research community was dedicated to elucidate the molecular processes of plant PCD and most characterization was based mainly on the morphological and ultrastructural features of dying
cells (Olvera-carrillo et al., 2015). Lesion mimic mutants (LMMs), i.e. plants that have dysregulated PCD, then became an important research tool in forward genetics aiding in the identification of biomolecules that are essential to PCD, chiefly in the form of hypersensitive response (HR; Lorrain, Vailleau, Balagué, and Roby, 2003).
Rice (Oryza sativa) LMM opened opportunities to study defense-related PCD in monocotyledonous plants, akin to Arabidopsis thaliana for dicots, and find genes and gene products that regulate this process.
HR, in particular, is essential in rice disease resistance as it limits the spread of the pathogen by sacrificing thru death the already infected cells. In brief, HR
occurs during an incompatible plant-pathogen interaction. This is a response at the early detection and recognition stage where a timely burst of reactive oxygen species (ROS) and other plant-derived toxic compounds are released at the site of infection. Due to this, pathogen invasion is slowed down and the host is given enough time to induce other plant defense responses including a PCD (Iakimova, Michalczuk, and Woltering, 2006). The molecular development leading to HR remains to be studied as there are several confusions on how it works and its overlapping roles in many other cellular processes not limited to the disease progression (Bashir et al., 2013). Fortunately, most LMMs exhibit an enhanced HR in the absence of pathogen and LMM genes are usually associated with plant defense signaling.
Studies on rice LMM have demonstrated increased resistance on important pathogens, including Magnaporthe oryzae, the causative agent of the serious rice blast (RB) disease with a devastating yield loss of more than 30% worldwide (Nalley, Tsiboe, Durand- Morat, Shew, and Thoma, 2016). Magnaporthe oryzae is a hemibiotrophic filamentous ascomycete that can elicit the host’s primary immune response via recognition of microbe-associated molecular patterns (MAMPs) such as chitin oligomers. Likewise, chitin elicitor binding proteins (CEBiP) of the host initiates the so-called the MAMP- triggered immunity (MTI) by successfully recognizing early events of pathogen invasion and launching an appropriate defense response. However, M. oryzae can evade this basal defense by releasing effector proteins that could lessen or shut down the signal generated by MTI and makes the host susceptible to an attack. For example, the fungus releases secreted LysM protein 1 (Slp1) that can competitively bind to CEBiP and reduce the signal necessary to start the cascade of defense reactions (Mentlak et al., 2012). To counter these pathogen-derived effectors, a matching resistance gene product should be present in the host, which will aid in recognizing the pathogen and allowing the plant to carry on the second line of defense called the effector-triggered immunity (ETI). This evolutionary “arms race” between plant and pathogen was the basis of the canonical zigzag model of the plant immune system (J. D. G. Jones and Dangl, 2006). More importantly, ETI produces a stronger defense-related response compared to MTI including said HR.
Several mutated rice lines with LMM phenotype have been successfully characterized. For instance, 21 LMMs generated from an indica rice variety (IR64) were established in the work of Wu et al. (2008). Further
studies on such LMMs identified important players of plant PCD; using rice LMM spotted leaf 11 (spl11), a non-functional U-Box/Armadillo Repeat Protein with an E3 ubiquitin ligase activity was found to negatively regulate PCD (Zeng et al., 2004). Similar studies on other plant PCD models showed LMM genes to have various functionality including fatty acid/lipid metabolism (Mou, He, Dai, Liu, and Li, 2000), biosynthesis of chlorophyll and heame (Ishikawa, Okamoto, Iwasaki, and Asahi, 2001), heat stress transcription factor (Yamanouchi, Yano, Lin, Ashikari, and Yamada, 2002) and as membrane-associated protein (Lorrain et al., 2004) among others.
A collaborative research between the Plant Pathology Laboratory of the International Rice Research Institute (IRRI), Laguna, Philippines and the State Key Laboratory of Hunan Hybrid Rice Research Center (HHRRC), Hunan, China, was conducted to characterize a LMM rice from the ethyl methanesulfonate (EMS)-generated mutant collection of IRRI. LMM 6321-2 displaying typical lesion mimicking phenotype with discrete and spontaneous lesions appearing at the late 5-leaves stage was used for further characterization. The study characterized the changes in important agronomic traits and RB resistance response between LMM 6321-2 and its indica cultivar parent, IR64. Also, a segregation analysis was used to determine the type of mutation in this LMM, followed by profiling the expression of selected genes involved in plant PCD to determine differentially expressed genes between LMM and IR64 with and without inoculation of incompatible RB isolate.
Materials and Methods
Plant Material. The mutated rice germplasm material was obtained from the Plant Pathology Laboratory, Genetics and Biotechnology Division at the International Rice Research Institute (IRRI), Philippines. The lesion mimic mutant rice was generated by ethyl methanesulfonate (EMS)-mediated mutagenesis in an indica IR64 background parent. A stable F4 generation of one of the mutants produced was generated and designated as IR64 6321-2 M4 herein referred to as LMM 6321-2. Control rice lines including IR64, CO39, LTH, and IRBL10 where also provided during resistance challenge test.
Measurement of Important Agronomic Traits. Forty plants of LMM 6321-2 and IR64 were established on 20 pots (two plants each pot) and maintained under
greenhouse conditions from November 2018 until mid- January 2019. At the start of the tillering stage, complete fertilizer was added, similar to the last stage of tillering.
After 110 days from transplantation, agronomic traits important for crop yield were measured. These included the plant height (PH; in cm; n = 20), number of tillers (NT; n = 20), number of panicle per plant (PPP;
n = 20), panicle length (PL; in cm; n = 50), number of primary branch per panicle (PBP; n = 50), seed setting rate (SSR), 1000 seed weight (SW; in g), grain yield per pot (GYP; in g; n = 20) and total leaf chlorophyll content (TCC). SSR were assessed by counting the number of filled (F) and unfilled (U) grains per panicle (n = 50).
SW were evaluated by measuring the weight of 1000 randomly selected filled grains in each pot (n = 10) and GYP was determined by measuring all collected filled seeds per pot (n = 10). Total leaf chlorophyll content was measured following the methods of Ritchie (2006).
Blast Inoculum Production and Plant Inoculation. The different Magnaporthe oryzae isolates were obtained from the Plant Pathology Department, Genetics and Biotechnology Division at IRRI, Philippines. Stock cultures of spores seeded in filter paper were revived using Prune Agar plates (PA; 3 pcs prune, 5 g α-lactose monohydrate, 21 g agar and 1 g yeast extract per liter) supplemented with 10 ppm streptomycin and then incubated at 30°C for 3-4 days.
The resistance challenge tests were conducted following IRRI protocol on RB inoculation (Hayashi, Kobayashi, Vera Cruz, and Fukuta, 2010). CA89 and PO6-6 were used as the virulent pathovars known to be compatible with the IR64 parent, while CA41, V86010 and M101-1-2-9-1 were used as the incompatible isolates known to induce HR in the LMM 6321-2 parent. Final concentration of spore suspension adjusted to 1.0 to 1.5 × 105 spores per ml (with 0.01% Tween 20) were collected from 3-4 days old cultures. Pregerminated seeds of each rice varieties including the parents IR64 and CO39, LMM 6321-2, LTH (susceptible control to all isolates) and IRBL10 (resistant control to CA89 and PO6-6) were sown on soil placed in plastic food keepers. Two methods of rice blast nursery (RBN) were employed, i.e. spraying method and punch inoculation.
For the spraying method, 14-day-old plants at the early 4-leaves stage were inoculated by around 20 ml of the different blast isolates through foliar spray of spore suspension. Set-up was placed in a dew chamber for 20-hours of incubation then transferred to a greenhouse with high humidity (mist room) at 25-30 ºC for seven days. For the punch inoculation method, 4-week-old plants were injured using a mouse clipper, inoculated with RB 20 μl conidial suspension, then
secured with clear tape. Observations and assessment were conducted 14 days post inoculation (dpi). Scoring for the infection phenotype was conducted using IRRI standards on RBN assessment on spray method where the experiment was conducted in triplicates per isolates with 10 (parents and controls) to 20 (mutant) plants per replicate (Hayashi et al., 2010). Scores ranging from zero through five were given and infection types of 0-2 were considered resistant to the pathogen while infection types 3-5 indicate susceptibility to the pathogen. On the other hand, percent disease lesion area per isolate per plant (n=15) was evaluated in the punched inoculated samples using ImageJ software.
Segregation analysis of the LMM 6321-2. The F2 population of LMM 6321-2 crossed to CO39 was also obtained from IRRI, Philippines. CO39 was used because it is susceptible to the incompatible RB isolates of IR64 and to successfully select segregating LMM lines. Five hundred seeds of the segregating population were prepared similar to the RBN assay. At 14 days after sowing, spore suspension of V86010 isolate was inoculated to the set-up, which included the parent IR64 as the negative control, M4 of LMM 6321-2 as the positive control and CO39 as an indicator or successful inoculation. After seven dpi, leaves were evaluated and the frequency of plant positive and negative to lesion response was recorded.
Expression Profiling of Cell Death Associated Genes. Both non-inoculated rice and RB inoculated rice were used for expression profiling. Rice plants were prepared as in RBN assay. The third leaf of IR64 and LMM 6321-2 was collected on the third (still no lesions observed) and fourth week (lesions starts to appear) for expression profiling study without inoculation. IR64, LMM 6321-2, CO39 and LTH were also prepared as in RBN assay then inoculated with M. oryzae V86010 (representative of incompatible interaction) to trigger cell death in the mutant. Leaf samples were collected at 0, 24 and 48 hours post inoculation (hpi) and stored at -80º C until RNA extraction. Total RNA collection was done using TaKaRa MiniBEST Plant RNA Extraction Kit (Takara Bio Inc., Beijing, China) then reverse transcribed using PrimeScript™
RT reagent Kit with gDNA Eraser (Takara Bio Inc., Beijing, China) following manufacturer’s protocol. The method used for the quantification was done following the protocol of Applied Biosystems 7300/7500 Real- Time PCR System using only half of the recommended reaction mixture using SYBR® as detection dye. Initial template denaturation before PCR was done at 95℃
for 30 sec., PCR reaction was done for 40 cycles at
95℃ for 5 sec and 60℃ for 34 sec, and the melting curve was performed at standard condition. Absolute quantification of each gene was conducted in triplicates and the relative expression level was obtained using the 2-ΔΔCt method. The expression of genes belonging to the same family of NADPH oxidases, representative ROS scavenging enzymes and previously characterized genes that played a central role in PCD together with genes that are crucial to phytohormone biosynthesis or regulation, pathogenesis-related proteins and senescence-associated genes were included during expression profiling.
Statistical Analysis. All data analyses were conducted using SPSS 23.0 software (IBM Corp.
Armonk, NY, USA). Measurements were presented as mean ± standard deviation. Comparisons between two groups were analyzed by independent sample t-test, while comparison among multiple groups were analyzed using one-way analysis of variance (ANOVA) with post-hoc analysis using Tukey HSD test to determine group clustering. When p-value < 0.05, result was considered statistically significant.
Results and Discussions
LMM 6321-2 was an initiator type of mutant
Lesion mimic mutants can be categorized depending on the type of lesions it produces. In general, they can be under an initiator class, which manifests lesions of discrete and variable size, and a propagation class, with runaway cell death upon trigger and lesion
that expands continuously (Moeder and Yoshioka, 2008). LMM 6321-2 used in this study spontaneously produced lesions at the late four-leaf stage of rice and were more severe in older leaves than in younger leaves. Additionally, lesions appeared to initiate at the tip of the leaves then multiplied and spread to the base as it grew older.
Mutation caused significant decrease in agronomic traits
To determine the penalty of dysregulated cell death on the agronomic characteristics of LMM 6321-2, various agronomic traits of the mutants were analyzed and compared with those of the IR64 parent.
The mutant generally exhibited reduced agronomic performance compared to the parental line (Table 1). There was a significant decrease in plant height, number of tillers per plant, number of panicles per plant, and panicle length. Additionally, the potential yield was also significantly reduced with decreased seed setting rate, 1000 seed weight, grain yield per pot and total chlorophyll content. Meanwhile, the number of primary panicle branches did not differ in both lines. Similar findings were observed by Xu et al.
(2014) with rice LMM spl30, which also showed lower trait values than wild-type. LMMs also generally have no beneficial effects on plant productivity as observed in other studied LMMs such as in wheat HLP mutant (Kamlofski et al., 2007), rice lmm6 (Xiao, Zhang, Lu, and Huang, 2015), rice spotted leaf sheath mutant (Lee et al., 2018) and rice llm1 (Wang et al., 2015). Furthermore, given that LMM 6321-2 was grown under greenhouse conditions, growing them in the field may still reduce agronomic performance as initially documented.
Table 1 Changes in agronomic traits in LMM 6321-1 compared to IR64 parent.
Agronomic Traits Mean ± SD
p-value*
IR64 LMM 6321-2
Plant Height (cm) 96.60 ± 3.10 86.44 ± 6.13 0.000
Number of Tillers per Plant 13.88 ± 3.26 11.50 ± 1.95 0.014
Number of Panicles Per Plant 13.81 ± 3.23 10.50 ± 1.54 0.001
Panicle Length (mm) 243.94 ± 23.63 235.32 ± 18.60 0.045
Number of 1° Panicle Branches 8.26 ± 1.05 8.36 ± 1.12 0.646
Seed Setting Rate (%) 79.61 ± 7.47 61.56 ± 9.02 0.000
1000 Seed Weight (g) 24.15 ± 1.09 20.15 ± 0.93 0.000
Grain Yield Per Pot (g) 45.28 ± 10.29 16.64 ± 1.09 0.000
Chlorophyll a + b content (μg/ml/g FW) 33.60 ± 3.15 26.11 ± 2.41 0.000
*Independent sample t-test significant at p < 0.05
LMM 6321-2 showed increased resistance against virulent M. oryzae pathovars
Most documented LMMs were found to have increased resistance to both biotic and abiotic stressors.
To determine changes in resistance of LMM 6321-2 against Philippine isolates of RB fungus, the mutant was inoculated with five different M. oryzae pathovars, two of which are virulent to the IR64 parent. Rice blast nursery (RBN) assay showed increased resistance of LMM 6321-2 to PO6-6 and partial resistance to CA89 (Fig. 1). Meanwhile, incompatible isolates to the IR64 parent triggered PCD in the mutant and showed severe lesion formation (Fig. 1) but no change in resistance response. Similarly, punch inoculated LMM 6321-2 leaves showed significantly decreased percent diseased lesion area in both PO6-6 and CA89 and statistically the same resistant response as IRBL10 (Fig. 2a and b).
As previously observed in RBN, the mutant showed more evident lesion formation in inoculated groups than negative control (0.01% Tween 20), indicating the ability of M. oryzae isolates to trigger cell death in LMM 6321-2.
Lesion formed in LMMs from altered PCD resembles a hypersensitive response (HR)-mediated
cell death that plays an essential role in plant immunity.
In the study of Wu et al. (2008), they found two diepoxybutane-mutagenized LMM rice, i.e. spl17 and Spl26, that conferred increased resistance to both blast fungus and bacterial blight (BB) pathogen. Similarly, previously cited works of Xiao et al. (2015) on lmm6, Lee et al. (2018) on rice spotted leaf sheath mutant and J. Wang et al. (2015) on rice llm1 all showed increased RB and BB resistance despite poorer agronomic performance. More importantly, LMMs on said works showed increased ROS production and significantly heightened expression of defense-related genes, which was associated with increased pathogen resistance.
Additionally, HR is frequently observed in an incompatible plant-pathogen interaction (Bagirova, 2007). Similar to LMM 6321-2 that had induced cell death after inoculation of incompatible isolates, Jung et al. (2005) also observed induction of chlorotic lesions on their LMM (blm) accompanied by a significant increase in phytoalexin production compared to the wild-type. Taken together, the enhanced cell death formation of LMM 6321-2 after blast inoculation may have contributed to its enhanced resistance against virulent pathovars.
Fig 1 Rice blast nursery assay on different rice lines compared to LMM 6321-2. The mutant showed increased resistance to PO6-6 and CA89, which was compatible to IR64 parent. Meanwhile, incompatible isolates V86010, M101-1-2-9-1 and CA41 triggered the lesion formation in the mutant with similar cell death observed when inoculated with a compatible blast isolate.
Fig 2 Resistance profile of punch inoculated blast on rice lines compared to LMM 6321-2 at 14 dpi. a) Disease reaction of different rice lines on various blast isolates showing smaller lesion area of LMM 6321-2 on virulent isolates and evident lesion induction compared to control.
b) Significantly decreased disease lesion area (%) of the mutant than IR64 parent and comparable resistance to control line IRBL10. Statistical significance is denoted by ** and *** where rice lines with different letters denote significantly different disease lesion area (%).
a
b
A single recessive gene caused LMM 6321-2
The mode of inheritance of LMM 6321-2 was studied using segregation analysis of its F2 population crossed with CO39. Using different incompatible blast isolates of the IR64 parent line but compatible with CO39, F2 generated LMM can be easily identified.
Chi-square analysis of each isolate showed that the mutation followed the expected 1:3 phenotype ratio of mutant to wild-type during a single, recessive mode of inheritance (Table 2). Fortunately, this means that the phenotype is most likely controlled by a change in only a single recessive gene, making it relatively easier to conduct further characterization.
PCD-associated genes in LMM 6321-2 were highly differentially expressed without pathogen inoculation
Three of the five oxidase genes, i.e. RbohB, RbohC and RbohD, were significantly different between IR64 and LMM 6321-2 (Fig. 3a). Meanwhile, representative ROS scavengers’ expressions significantly changed where OsAPX was reduced at week 3 when three NADPH oxidases were highly expressed (Fig. 3b). Understandably, OsCATb and cytCuZnSOD1 were found differentially expressed in LMM 6321-2 probably to negate the ROS burst. All LMM-associated genes tested were also significantly upregulated in the mutant during week 3 then decreased at week 4 (Fig.
3c). Furthermore, three of the phytohormone-related genes were also significantly heightened during week 3, including OsYUCCA for auxin biosynthesis, OsNPR1 as salicylic acid signaling receptor and OsACS2 for ethylene biosynthesis (Fig. 3d). Only one of three defense response indicators was highly expressed in LMM 6321-2, i.e. PR5 and only during the 3rd week (Fig.
3e). Lastly, all four senescence indicators were found highly expressed on week 3 in the mutant, while only three (OsSAG-12, Osh69 and Osh36) were significantly upregulated (Fig. 3f). Remarkably, the 4th leaf at week 3 showed no signs of lesions. However, since phenotype
was observed in an age-dependent manner (and 3rd leaf had visible lesions at week 3), it can be inferred that the 4th leaf was already undergoing some dramatic changes in its expression pattern. Taken together, the LMM 6321-2 expression profile was significantly different from the IR64 parent where LMM 6321-2 mutation could affect the global expression of several genes.
LMM 6321-2 exhibited delayed expression response upon incompatible blast inoculation
To determine changes in gene expression status of LMM 6321-2 during its early phases, an incompatible blast isolate was used to trigger the lesion mimic phenotype using RBN. Three of the NADPH oxidases were also found to have significantly different expression profiles, with OsRbohC being the most highly expressed in both IR64 and LMM 6321-2 peaking at 24 hpi for IR64 and 48 hpi for LMM 6321-2 (Fig. 4a).
On the other hand, both IR64 and LMM 6321-2 have a similar OsRbohA expression trend, which heightened at 48 hpi, but the relative expression for IR64 was significantly higher than LMM 6321-2. A different pattern was observed with OsRbohE in both parent and mutant, with expression significantly downregulated at 24 hpi and increased significantly at 48 hpi. All three ROS scavengers tested were found to have significantly different expression profiles with OsAPX1 and OsCATb of IR64 following the same expression as OsRbohC, which significantly peaks at 24 hpi (Fig. 4b). The same significant increase was noted with cytCuZnSOD1 of IR64, but the change was minimal. Understandably, ROS homeostasis is normally kept in check by balancing the expression and activity of ROS producers and scavengers. The same observations were noted with LMM 6321-2 where OsAPX1 and cytCuZnSOD1 peaked at 48 hpi together with its corresponding OsRboh genes.
Interestingly, the expression of the tested catalase gene OsCATb did not differ significantly across all time points though a minimal increase was noted at 24 hpi.
This imbalance in ROS producers’ and ROS scavengers’
expression in LMM 6321-2 may be the key difference Table 2 Chi-Square test for segregation of lesion mimicking F2 population (CO39 × IR64-6123 M1-1) inoculated with different incompatible blast isolates.
Blast Isolate Total Number of Plants
Observed Lesion Mimic Positive Lesion Mimic
Negative χ2 P-value*
V86010 151 44 107 1.38 0.240
CA41 127 35 92 0.44 0.505
M101-1-2-9-1 186 50 136 0.35 0.553
*The goodness of fit to a 1:3 ratio is indicated when p > 0.05
Fig 3 Expression profiles of PCD-related gene expression in non-inoculated LMM 6321-2 and IR64. Normalized expression levels of a) a family of Respiratory Burst Oxidase Homologs (RBOH), b) ROS scavenger enzymes, c) LMM-related genes, d) phytohormone-related genes e) defense response indicators and f) senescence associated genes collected during third (W3) and fourth week (W4). Data were expressed as mean ± SD of quantified genes by qRT-PCR normalized using 18S rRNA. Statistical significance was determined using Welch test and was labeled significant when *p < 0.05, **p < 0.01 and ***p < 0.0001. Different letters denote statistically different expression levels between rice lines and age using Games-Howell post-hoc test.
a
c
e
b
f d
Fig 4 Expression profiles of PCD-related gene expression in blast-inoculated LMM 6321-2 and IR64. Relative expression levels of a) a family of Respiratory Burst Oxidase Homologs (RBOH), b) ROS scavenger enzymes, c) LMM-related genes, d) phytohormone-related gene and e) defense response indicators during 0, 24 and 48 hpi. Data were expressed as mean ± SD of quantified genes by qRT-PCR normalized using 18S rRNA. Statistical significance was determined using One-way ANOVA and was labeled significant when *p < 0.05, **p < 0.01 and ***p <
0.0001. Different letters denote statistically different expression levels between rice lines and sampling time points using Tukey HSD post-hoc test.
a
c
d
b
e
in the induction of HR-mediated PCD compared to its parent.
Only three of the five LMM-related genes produced quality qRT-PCR reads in this experiment, but all of them produced significantly different expression profiles when IR64 and LMM 6321-2 were compared (Fig. 4c). For IR64, the same observations were noted in the expressions of OsRac1, OsLSD1 and OsSpl11 with significant upregulations at 24 hpi then downregulated at 48 hpi. Similarly, all LMM-associated genes in LMM 6321-2 were significantly upregulated only later at 48 hpi with 0 and 24 hpi relatively having the same expression levels.
Previously used genes in the non-inoculated experiment that controls the rate-limiting steps or important proteins in phytohormone biosynthesis (OsNCED1, OsJAR1 and OsYUCCA1 among others) did not produce quality qRT-PCR results in the current experiment. Similar to the case of LMM-related genes, expression of these genes may be below the detection capacity of the current kit that was used. Another gene, OsICS1, which controls the production of salicylic acid precursors were instead tested. A similar expression pattern as those in NADPP oxidases among others was observed in IR64 with OsICs1 expression significantly elevated at 24 hpi then dropped back after 48 hpi (Fig.
4d). Meanwhile, the expression of OsICS1 in LMM 6321-2 increased after 24 hrs and then sustained until 48 hpi.
Since LMM 6321-2 was found to have partial resistance against compatible blast isolates, it was hypothesized that defense response could be either constitutively expressed or easily inducible to attain resistant phenotype. Interestingly, two of the working pathogenesis-related (PR) genes used in this experiment, OsPR1a and OsPR5, showed significantly lower expression at early time points and followed previously observed trends i.e. increasing at 48 hpi (Fig. 4e). Though an incompatible isolate was used in this experiment, it can be inferred that the resistant phenotype was not attributed to either hypothesis mentioned; instead, it was more likely that LMM 6321- 2 produces a delayed but sustained immune response.
Meanwhile, OsPR5 of IR64 could play an important role in defense response against blast fungus. It coincided with other PCD-related genes, which increases at 24 hpi compared to OsPR1a that have initially high expression then significantly downregulated as hours post inoculation progresses.
Conclusions and Recommendations
Lesion-mimicking mutant (LMM) 6321-2 showed penalty of an induced PCD via reduced agronomic potential. However, this caused LMM 6321-2 to have increased and partial resistance to compatible M. oryzae isolates while incompatible Philippine RB isolates were able to trigger and induce its lesion formation without changing disease resistance. Similar to previous works on LMMs, these findings suggest a possible HR-related PCD in the mutant due to the increased resistance to virulent blast fungus isolates. The mode of inheritance of the mutated gene causing the phenotype was also established to be caused by a single recessive genetic mutation, allowing this mutated line to be a good model organism for future studies to elucidate novel key players in plant PCD. Preliminary expression profiling also showed a global change in HR-PCD related pathways, which partially explains the increased resistance to compatible RB isolates primarily through a sustained, though a late expression of HR-PCD related genes. With this, it is highly recommended that further molecular work be done on LMM 6321-2, such as the use of bulk segregation analysis to pinpoint the location of the mutation within the entire genome of LMM 6321-2 and also a transcriptomic study to determine other dysregulated pathways brought about by the mutation.
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Recommended citation:
Paet, J.M.Q. et al. (2022). Rice Lesion Mimic Mutant (LMM) 6321-2 Exhibits Resistance Against Rice Blast (Magnaporthe oryzae) via Increased Hypersensitive Response. Bicol University Research & Development (BUR&D) Journal. 25 (1), 40-50. doi: 10.47789/burdj.
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