Rice is the dominant staple crop in the world, and the demand for rice production is still rising with the increasing population. However, rice blast caused by the fungus (M. grisea
) leads to the decrease of rice yields widely and destructively, threatening global food security [1
]. Rice leaves, nodes, collars, panicles, and roots are easily infected by the pathogen at all growth stages [3
]. Pesticides have been over-applied to control rice blast, resulting in globe pollution. Therefore, the use of rice cultivars with resistant ability to blast fungus has been regarded as the most economical and environmental friendly approach to control this disastrous disease [4
]. However, many rice cultivars only conferred race-specific resistance, and their resistance was short-lived, presumably due to strong selection pressure for high pathogenic variability of the fungus [7
Isolating and subsequently identifying resistant (R) genes from rice would help to elucidate multiple molecular mechanisms for host resistance and pathogen virulence. Over 100 R genes were mapped on the rice genome, and 35 of them have been cloned [9
]. However, the rice resistant ability with the single R gene is not durable due to pathogen evolution. Breeders aim to develop new rice varieties, which could balance durable resistance with yield. Deng et al. reported that two genes, including PigmR
, on one locus worked together to confer broad-spectrum resistance to blast fungus M. grisea
without yield penalty [10
]. Li et al. identified a natural allele of a transcription factor in rice with a non-race specific resistant ability to blast that carried no observable penalty in plant growth or yield [11
]. Wang et al. revealed that a transcription factor, Ideal Plant Architecture 1
), could regulate the expression of DEP1
through phosphorylation to balance resistance with yield [12
]. Although the classical gene-for-gene theory could partially account for rice race-specific resistant ability to M. grisea
], only seven avirulence genes (Avr) in M. grisea
corresponding to R genes were studied, including Pi-ta
], and Pii
Proteomics, one of the most important research methods in the post-genomics era, has been widely used to study the plant response mechanism to various stresses, especially diseases. Proteomics analysis of resistant and susceptible soybean varieties infected by Phytophthora sojae
indicated that 30 and 20 differential expression proteins (DEPs) related to metabolism, energy regulation and protein storage, and degradation were discovered in a resistant and susceptible line, respectively [20
]. After Xanthomonas oryzae
infection, 32 proteins concerned with signal transduction, pathogenesis, and cell metabolism were up-regulated in rice leaves, and 23 of them were supported by the microarray data [21
]. Ventelon-Debout et al. found 40 and 24 DEPs existed in resistant and susceptible rice varieties in response to the infection of rice yellow mettle virus (RYMV), respectively [22
However, the relationship between the expression level of blast-induced proteins and the resistant ability of rice plants in response to the infection of different blast fungal isolates was not fully illuminated. In this study, proteomic methods were employed to analyze the DEPs in three rice varieties, which share the same genetic background, but show different resistant abilities, after inoculation with two different virulence isolates for 24 and 48 hours. The analysis on the clustering and functional module of DEPs revealed the different response mechanism of rice plants to different fungal isolates.
C101PKT (CN-4a) and C105TTP-4L-23 (CN-4b) with single blast resistant genes were developed by backcrossing resistant donor rice cultivars, Pai-Kan-Tao and Tetep, to the recurrent parent, CO39, respectively. Furthermore, the genetic studies showed that the resistant gene in CN-4a is allelic to that in CN-4b [23
]. The pathogen inoculation results showed that both of the two NILs were resistant to blast isolate 81278ZB15, and only CN-4b was resistant to blast isolate GUY11, whereas CO39 was susceptible to both of the two blast isolates. The previous research reported that these two fungal isolates had different virulence [24
]. Thus, in this study, these three rice varieties with different blast fungus resistant abilities and the two fungal isolates with different virulence were employed as materials to reveal the protein expression pattern in rice in response to different blast isolates’ infection by comparing their DEPs.
A total of 50 proteins in CO39 and its two NILs were found to be up-regulated in response to the two blast isolates’ infection. Thirteen proteins were reported to be induced by M. grisea
in a previous research work [25
], including heat shock 70 kDa protein (spot 4), NADH dehydrogenase subunit G (spot 12), NADP-dependent malic enzyme (spot 15), class III peroxidase (spot 24), glutathione S-transferase (spot 30), guanine nucleotide-binding protein subunit β-like protein (spot 31), photosystem II oxygen-evolving complex protein 1 (spot 33), L-ascorbate peroxidase (spot 36), chitinase (spot 39), dehydratase family protein (spot 40), β-glucanase (spot 41), photosystem II oxygen-evolving enhancer protein 2 (spot 44), and dihydrolipoamide dehydrogenase family protein (spot 46). Sasaki et al. also found that class III peroxidase (spot 24) was up-regulated vastly after the infection of blast fungus and its high expression was related to the defense reaction, the response to blast fungus, and basal resistance [28
]. Nishizawa et al. confirmed that the up-regulation and constitutive expression of chitinase (spot 39) enhanced rice resistant ability to blast fungus [29
]. Nakashima et al. revealed that guanine nucleotide-binding protein subunit β-like protein (spot 31) was up-regulated and interacted with the Rac 1 immune protein complex to improve blast resistant ability for rice [30
]. However, some DEPs might be the key molecule in rice to resist blast fungus, but this not yet been reported previously. For instance, ferritin (spot 43) in CN-4b was up-regulated at 24 hpi, which was the critical response time for rice plants to resist blast fungus [31
plants with ferritin knocked out were sensitive to excessive iron, were deficient in growth and flowering, and showed an accumulation of the reactive oxide group [32
]. Therefore, we believe that the increasing expression level of ferritin in CN-4b at 24 hpi might balance the plant growth and its resistance to fungus.
A total of 21 proteins (spots 2, 3, 5, 7–9, 16, 20, 25–27, 29, 30, 32, 37, 39, 40, 43, 44, 47) were up-regulated in response to 81278ZB15 infection and 23 proteins (spots 2, 4, 7, 14, 16, 18–20, 23–27, 31–32, 38–39, 41, 43–45, 48–49) were induced to increase in response to GUY11 infection (Table S1
). It could be inferred that those 21 and 23 proteins were related to the resistance capacity against 81278ZB15 and GUY11, respectively. Moreover, six proteins-spots 2, 20, 32, 16, 25, and 26 (Table S1
) in CN-4b were up-regulated at 24 and 48 hpi of both two blast isolates, respectively, hinting that these proteins might contribute to a broad-spectrum resistant ability in CN-4b to blast fungus.
Clustering analysis on the expression pattern of DEPs has been widely used to find a related expression module. Salekdeh et al. discovered that protein clusters in different rice varieties’ response to drought stress in the same expression pattern and then they revealed the identical hereditary in rice in response to drought [33
]. In this research, DEPs’ expression levels were clustered exactly into two branches corresponding to the two pathogenic isolates, indicating that DEPs’ cluster could reflect the race-specific resistant ability in rice. Meanwhile, compared to the other two varieties, DEPs in CN-4b at 24 and 48 hpi of both two blast isolates were always clustered together with a characteristic module, inferring that CN-4b had a unique basal defense system to blast fungus.
The functional modules of DEPs were grouped into two branches correlated to the two pathogenic isolates. This result was consistent with that in the DEP cluster analysis. The photosynthesis-related module ME 3 in resistant varieties (CN-4a and CN-4b) was expressed higher than that in the susceptible one CO39 in response to the infection of 81278ZB15, because its photosynthesis was sensitive to various stresses and it decreased after pathogen infection [34
]. When the plant was infected by pathogens, free radicals and excessive peroxides were produced and this resulted in damage to cells [35
]. The increasing expression level of oxidative stress (ME 4 and 6) in CN-4b might confer resistance to GUY11 by removing the free radicals and regulating peroxide accumulation [11
In summary, 50 proteins were found to be up-regulated in rice in response to different fungal isolate infection, and the expression pattern and functional modules of these 50 DEPs were clustered into two branches corresponding to the two blast isolates, indicating that different DEPs contribute to the race-specific resistant ability in rice.